Tetrahydroisoquinolines as PRMT5 inhibitors

ABSTRACT

A compound of formula (I) wherein: n is 1 or 2; p is 0 or 1; R 1a , R 1b , R 1c  and R 1d  are independently selected from the group consisting of H, halo, C 1-4  alkoxy, C 1-4  alkyl, C 1-4  fluoroalkyl, C 3-4  cycloalkyl, NH—C 1-4  alkyl and cyano; R 2a  and R 2b  are independently selected from the group consisting of: (i) F; (ii) H; (iii) Me; and (iv) CH 2 OH; R 2c  and R 2d  are independently selected from the group consisting of: (i) F; (ii) H; (iii) Me; and (iv) CH 2 OH; R 2e  is H or Me; R 3a  and R 3b  are independently selected from H and Me; R 4  is either H or Me; R 5  is either H or Me; R 6a  and R 6b  are independently selected from H and Me; A is either (i) optionally substituted phenyl; (ii) optionally substituted naphthyl; or (iii) optionally substituted C 5-12  heteroaryl; wherein when R 2e  is H, at least one of R 1a , R 1b , R 1c  and R 1d  is selected from C 1-4  alkoxy, C 2-4  alkyl, C 1-4  fluoroalkyl, C 3-4  cycloalkyl, NH—C 1-4  alkyl and cyano.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a § 371 National State Application ofPCT/EP2017/055529 filed Mar. 9, 2017 which claims priority to GB1604020.6 filed Mar. 9, 2016.

The present invention relates to C-alkyl bicyclic amines and their useas pharmaceuticals, and in particular, in treating cancer andhemoglobinopathies.

BACKGROUND TO THE INVENTION

Post-translational modification of proteins is a hallmark of signaltransduction where cells are able to react quickly to changes or eventsin the cellular environment. Post-translational modification of proteinsexpands the structural and functional diversity of the proteome. Therole of acetylation and phosphorylation of proteins has been extensivelystudied as highly reversible reactions for fine-tuning responses toexternal stimuli or changes in the environmental conditions. Recently,the importance of other types of protein modifications, includingubiquitination and methylation has begun to be recognized.

The methylation of proteins and the enzymes that carry out thesereactions has increased the dimensions of gene regulation by markinggenes that are transcriptionally active or silenced. Protein methylationcan occur on amino acids such as lysine, arginine, histidine, orproline, and on carboxy groups.

Arginine methylation of mainly nuclear proteins is an importantpost-translational modification process involved in structuralremodelling of chromatin, signal transduction, cellular proliferation,nucleocytoplasmic shuttling, translation, gene transcription, DNArepair, RNA processing, or mRNA splicing.

Methylation of proteins at arginine residues is catalysed by ProteinArginine Methyltransferase enzymes. The Protein Arginine MethylTransferase (PRMT) family of enzymes are evolutionarily conservedbetween organisms but differ in the number of members in differentorganisms.

There are eleven members of the human PRMT family, eight of which haveknown enzymatic activity and target substrates. With the exception ofPRMT2 and two recently identified putative PRMT genes (PRMT10 andPRMT11), all remaining proteins of the family possess enzymatic argininemethylation activity.

PRMTs are subdivided into two types based on the methylation that theycatalyse at the guanidinium group of arginine residues of substrateproteins. There are three nitrogens in the guanidinium group,potentially all of which could be methylated; the two ψ-guanidinonitrogen atoms and the internal δ-guanidino nitrogen atom.Mono-methylation and dimethylation of arginine (MMA and DMA) is found inmammalian cells at one or both of the two ψ-guanidino nitrogen atoms;dimethylation may be either symmetric or asymmetric. The thirdmethylated arginine is generated by monomethylation of the internalδ-guanidino nitrogen atom of arginine and has so far been documentedonly in yeast proteins. Type I PRMT enzymes catalyse the formation ofMMA and asymmetric dimethylarginine by di-methylating the same nitrogenatom of the guanidinium group, whereas Type II PRMT enzymes catalyse theformation of MMA and symmetric di-methylarginine by mono-methylatingeach of the terminal nitrogen atoms. Type III enzymes methylate theinternal δ-guanidino nitrogen atom. Of the eight well characterisedhuman PRMTs, PRMT1, 3, 4, 6 and 8 are Type I enzymes, and PRMT5, 7 and 9are Type II enzymes.

PRMTs catalyse the methylation of the guanidino nitrogen atoms ofarginine residues through the transfer of a methyl group from S-adenosylmethionine (SAM). A by-product of the enzymatic methylation step isS-adenosyl-L-homocysteine (AdoHcy), which is hydrolyzed to adenosine andhomocysteine by AdoHcy hydrolase (Krause et al., 2007).

PRMT5

PRMT5 (aka JBP1, SKB1, IBP72, SKB1his and HRMTIL5) is a Type II argininemethyltransferase, and was first identified in a two-hybrid search forproteins interacting with the Janus tyrosine kinase (Jak2) (Pollack etal., 1999).

PRMT5 plays a significant role in control and modulation of genetranscription. Inter alia, PRMT5 is known to methylate histone H3 atArg-8 (a site distinct from that methylated by PRMT4) and histone H4 atArg-3 (the same site methylated by PRMT1) as part of a complex withhuman SWI/SNF chromatin remodelling components BRG1 and BRM.

In addition to direct repressive histone marks induced by PRMT5, theenzyme's role in gene silencing is also mediated through the formationof multiprotein repressor complexes that include NuRD components, HDACs,MDB proteins and DNA methyltransferases, (Rank et al., 2010; LeGuezennec et al., 2006; Pal et al., 2003).

PRMT5 is involved in the methylation and functional modulation of thetumour suppressor protein p53. See (Berger, 2008; Durant et al., 2009;Jansson et al., 2008; Scoumanne et al., 2009). Most of the physiologicalfunctions of p53 are attributable to its role as a transcriptionalactivator, responding to agents that damage DNA. p53 status is wild typein approximately half of human cancer cases. These include 94% incervix, 87% in blood malignancies, 85% in bones and endocrine glands,and 75% of primary breast cancer. Restoration of p53 in cancer cellsharbouring wild type p53, by way of inhibiting mechanisms that suppressits function leads to growth arrest and apoptosis, and is regarded as apotentially effective means of tumour suppression.

p53 target genes have two alternative downstream effects: either theypause the cell cycle, allowing the DNA to be repaired, or, if repair isnot possible, they activate processes leading to apoptosis (programmedcell death). How p53 ‘chooses’ between these distinct outcomes is acentral question in the field of tumour biology.

p53 is replete with post-translational modifications. Phosphorylationwas one of the first post-translational modifications to be clearlydefined on p53. In the last decade it has become additionally clear thatp53 is modified not only by phosphorylation, but that it is extensivelymodified by lysine acetylation and methylation, among othermodifications. Indeed, besides histone proteins p53 is the most commonprotein substrate known for these post-translational modifications.However, despite the plethora of post-translational modifications, p53has not been identified, until recently, as a substrate for argininemethylation.

Jansson et al (Jansson et al., 2008) discovered that PRMT5 is physicallyassociated with a p53 cofactor called Strap. A co-factor complex thatcontains Strap et al binds to p53 in response to DNA damage. Jansson etal demonstrated that PRMT5 methylates p53 in vitro, and mapped the sitesof methylation (R333, R335 and R337). They developed an antibody thatspecifically detects p53 methylated on these sites and confirmed thatp53 is methylated in vivo. Jansson et al went on to show that p53methylation requires PRMT5 and is increased in response to etoposide, aDNA damaging agent.

The role of PRMT5 and p53 arginine methylation on cell cycle regulationand DNA damage response have been explored by both Jansson et al andScoumanne et al (Jansson et al., 2008; Scoumanne et al., 2009). Althoughsome differences are evident between the results from the two groups inrespect of cell cycle regulation in unperturbed cells (which may beascribed to cell type specific effects and/or the actual nature of theexperimental arrangements), both groups report similar results withrespect to the DNA damage response.

In response to DNA damage, caused by a variety of agents, includingdoxorubicin, camptothecin and UV light, and also in response totreatment with Nutlin-3, knockdown of PRMT5 results in an increase insub-G1 population and concomitant reduction in G1 cells and, in thepresence of p53, a significant increase in apoptosis. Knockdown of PRMT5also resulted in a reduced level of p21, a key p53 target gene thatregulates cell cycle arrest during the p53 response and MDM2, a p53 E3ubiquitin ligase, but not PUMA, NOXA, AIP1 & APAF1, p53 target geneslinked to apoptosis.

Knockdown of PRMT5 (but not PRMT1 or CARM1/PRMT4) results in decreasedp53 stabilisation, decreased basal p53 levels, and decreased p53oligomerisation, and also decreased expression of eIF4E a majorcomponent of translational machinery involved in ribosome binding tomRNA. Indeed, eIF4E is a potent oncogene, which has been shown topromote malignant transformation in vitro and human cancer formation.

Knockdown of PRMT5 would be expected to lead to a reduction in the levelof arginine methylated p53. Consistent with arginine methylation statusof p53 influencing the p53 response (reduced arginine methylationbiasing the response to proapoptotic), Jannson et al showed that a p53mutant in which each of the three critical arginine residues weresubstituted with lysine (p53KKK) retained the ability to induceapoptosis but its cell cycle arrest activity was significantlycompromised.

Moreover, pS3KKK also has a significantly reduced ability to inducetranscription of p21, by contrast with APAF1. The promoter bindingspecificity of wild-type p53 to key target genes is also significantlyaffected by arginine methylating status: Knockdown of PRMT5 results indecreased p53 binding to the promoter regions of the p21 and(intriguingly) PUMA genes, but does not affect p53 binding to thepromoter regions of NOXA or APAF1.

Taken together, it would seem that PRMT5 is a pro-survival factor, whichregulates cell proliferation in unstressed conditions and modulates thep53 response during DNA damage. In particular, knockdown of PRMT5,leading to a reduction in the levels of arginine methylated p53, appearsto bias the p53 DNA damage response to proapoptotic as opposed to cellcycle arrest.

PRMT5 is further linked to cancers in that it is aberrantly expressed inaround half of human cancer cases. PRMT5 overexpression has beenobserved in patient tissue samples and cell lines of Prostate cancer (Guet al., 2012), Lung cancer (Zhongping et al., 2012), Melanoma cancer(Nicholas et al., 2012), Breast cancer (Powers et al., 2011), Colorectalcancer (Cho et al., 2012), Gastric cancer (Kim et al., 2005), Esophagusand Lung carcinoma (Aggarwal et al., 2010) and B-Cell lymphomas andleukemia (Wang, 2008). Moreover, elevated expression of PRMT5 inMelanoma, Breast and Colorectal cancers has been demonstrated tocorrelate with a poor prognosis.

Lymphoid malignancies including CLL are associated with over-expressionof PRMT5. PRMT5 is over-expressed (at the protein level) in the nucleusand cytosol in a number of patient derived Burkitt's lymphoma; mantlecell lymphoma (MCL); in vitro EBV-transformed lymphoma; leukaemia celllines; and B-CLL cell lines, relative to normal CD19+ B lymphocytes (Palet al., 2007; Wang et al., 2008). Intriguingly, despite elevated levelsof PRMT5 protein in these tumour cells, the levels of PRMT5 mRNA arereduced (by a factor of 2-5). Translation of PRMT5 mRNA is however,enhanced in lymphoma cells, resulting in increased levels of PRMT5 (Palet al., 2007; Wang et al., 2008).

In addition to genomic changes, CLL, like almost all cancers, hasaberrant epigenetic abnormalities characterised by globalhypomethylation and hot-spots of repressive hypermethylation ofpromoters including tumour suppressor genes. While the role ofepigenetics in the origin and progression of CLL remains unclear,epigenetic changes appear to occur early in the disease and specificpatterns of DNA methylation are associated with worse prognosis (Chen etal., 2009; Kanduri et al., 2010). Global symmetric methylation ofhistones H3R8 and H4R3 is increased in transformed lymphoid cell linesand MCL clinical samples (Pal et al., 2007), correlating with theoverexpression of PRMT5 observed in a wide variety of lymphoid cancercell lines and MCL clinical samples.

PRMT5 is therefore a target for the identification of novel cancertherapeutics.

PRMT5 Function and Hemoglobinopathies

Hemoglobin is a major protein in red blood cells and is essential forthe transport of oxygen from the lungs to the tissues. In adult humans,the most common hemoglobin type is a tetramer called hemoglobin A,consisting of two α and two β subunits. In human infants, the hemoglobinmolecule is made up of two α and two γ chains. The gamma chains aregradually replaced by subunits as the infant grows. The developmentalswitch in human ß-like globin gene subtype from foetal (γ) to adult (ß)that begins at birth heralds the onset of the hemoglobinopathiesß-thalassemia and sickle cell disease (SCD). In ß-thalassemia the adultchains are not produced. In SCD a point mutation in the coding sequencein the ß globin gene leads to the production of a protein with alteredpolymerisation properties. The observation that increased adult γ-globingene expression (in the setting of hereditary persistence of foetalhemoglobin (HPFH) mutations) significantly ameliorates the clinicalseverity of ß-thalassemia and SCD has prompted the search fortherapeutic strategies to reverse γ-globin gene silencing. To date, thishas been achieved through pharmacological induction, using compoundsthat broadly influence epigenetic modifications, including DNAmethylation and histone deacetylation. The development of more targetedtherapies is dependent on the identification of the molecular mechanismsunderpinning foetal globin gene silencing. These mechanisms haveremained elusive, despite exhaustive study of the HPFH mutations, andconsiderable progress in many other aspects of globin gene regulation.

PRMT5 plays a critical role in triggering coordinated repressiveepigenetic events that initiate with dimethylation of histone H4Arginine 3 (H4R3me2s), and culminate in DNA methylation andtranscriptional silencing of the γ-genes (Rank et al., 2010). Integralto the synchronous establishment of the repressive markers is theassembly of a PRMT5-dependent complex containing the DNAmethyltransferase DNMT3A, and other repressor proteins (Rank et al.,2010). DNMT3A is directly recruited to bind to the PRMT5-inducedH4R3me2s mark, and loss of this mark through shRNA-mediated knock-downof PRMT5, or enforced expression of a mutant form of PRMT5 lackingmethyltransferase activity leads to marked upregulation of γ-geneexpression, and complete abrogation of DNA methylation at theγ-promoter. Treatment of human erythroid progenitors with non-specificmethyltransferase inhibitors (Adox and MTA) also resulted inupregulation of γ-gene expression (He Y, 2013). Inhibitors of PRMT5 thushave potential as therapeutics for hemoglobinopathies such asß-thalassemia and Sickle Cell Disease (SCD).

The present inventors have developed particular substituted β-hydroxyamides inhibit the activity of PRMT5 and therefore may be of use intreating conditions ameliorated by the inhibition of the activity ofPRMT5.

SUMMARY OF THE INVENTION

A first aspect of the present invention provides a compound of formulaI:

wherein:

n is 1 or 2;

p is 0 or 1;

R^(1a), R^(1b), R^(1c) and R^(1d) are independently selected from thegroup consisting of H, halo, C₁₋₄ alkoxy, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl,C₃₋₄ cycloalkyl, NH—C₁₋₄ alkyl and cyano;

R^(2a) and R^(2b) are independently selected from the group consistingof:

-   -   (i) F;    -   (ii) H;    -   (iii) Me; and    -   (iv) CH₂OH;

R^(2c) and R^(2d) are independently selected from the group consistingof:

-   -   (i) F;    -   (ii) H;    -   (iii) Me; and    -   (iv) CH₂OH;

R^(2e) is H or Me;

R^(3a) and R^(3b) are independently selected from H and Me;

R⁴ is either H or Me;

R⁵ is either H or Me;

R^(6a) and R^(6b) are independently selected from H and Me;

A is either

-   -   (i) optionally substituted phenyl;    -   (ii) optionally substituted naphthyl; or    -   (iii) optionally substituted C₅₋₁₂ heteroaryl;

wherein when R^(2e) is H, at least one of R^(1a), R^(1b), R^(1c) andR^(1d) is selected from C₁₋₄ alkoxy, C₂₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄cycloalkyl, NH—C₁₋₄ alkyl and cyano.

A second aspect of the present invention provides a compound of thefirst aspect for use in a method of therapy. The second aspect alsoprovides a pharmaceutical composition comprising a compound of the firstaspect and a pharmaceutically acceptable excipient.

A third aspect of the present invention provides a method of treatmentof cancer, comprising administering to a patient in need of treatment, acompound of the first aspect of the invention or a pharmaceuticalcomposition of the first aspect of the invention. The third aspect ofthe present invention also provides the use of a compound of the firstaspect of the invention in the manufacture of a medicament for treatingcancer, and a compound of the first aspect of the invention orpharmaceutical composition thereof for use in the treatment of cancer.

As described below, the compound of the first aspect may be administeredsimultaneously or sequentially with radiotherapy and/or chemotherapy inthe treatment of cancer.

A fourth aspect of the present invention provides a method of treatmentof hemoglobinopathies, comprising administering to a patient in need oftreatment, a compound of the first aspect of the invention or apharmaceutical composition of the first aspect of the invention. Thefourth aspect of the present invention also provides the use of acompound of the first aspect of the invention in the manufacture of amedicament for treating hemoglobinopathies, and a compound of the firstaspect of the invention or pharmaceutical composition of the firstaspect of the invention for use in the treatment of hemoglobinopathies.

Definitions

C₅₋₁₂ heteroaryl: The term “C₅₋₁₂ heteroaryl” as used herein, pertainsto a monovalent moiety obtained by removing a hydrogen atom from anaromatic structure having from 5 to 12 rings atoms, of which from 1 to 3are ring heteroatoms. The term ‘aromatic structure’ is used to denote asingle ring or fused ring systems having aromatic properties, and theterm ‘ring heteroatom’ refers to a nitrogen, oxygen or sulphur atom.

In this context, the prefixes (e.g. C₅₋₁₂, C₅₋₆, etc.) denote the numberof atoms making up the aromatic structure, or range of number of atomsmaking up the aromatic structure, whether carbon atoms or heteroatoms.

Examples of C₅₋₁₂ heteroaryl structures include, but are not limited to,those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆); pyridone (C₆); indole(C₉); quinoline (C₁₀);

O₁: furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅);

N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅);

N₁S₁: thiazole (C₅), isothiazole (C₅);

N₂S₁: thiadiazole (C₅)

N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆); benzimidazole(C₉)

N₃: triazole (C₅), triazine (C₆).

Cyano: The term “cyano” as used herein pertains to a group —CN.

Optional Substituents

The optional substituents for the phenyl, naphthyl and C₅₋₁₂ heteroarylgroups in A may be selected from the following groups.

C₁₋₄ alkyl: The term “C₁₋₄ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a saturated hydrocarbon compound having from 1 to 4 carbonatoms.

Examples of saturated alkyl groups include, but are not limited to,methyl(C₁), ethyl(C₂), propyl(C₃), and butyl(C₄).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl(C₁), ethyl(C₂), n-propyl(C₃), and n-butyl(C₄).

Examples of saturated branched alkyl groups include iso-propyl(C₃),iso-butyl(C₄), sec-butyl(C₄) and tert-butyl(C₄).

The term “C₂₋₄ alkyl” as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from a carbon atom of a saturatedhydrocarbon compound having from 2 to 4 carbon atoms.

C₁₋₄ fluoroalkyl: The term “C₁₋₄ fluoroalkyl” refers to a C₁₋₄ alkylgroup as defined above where one of more of the hydrogen atoms isreplaced by a fluoro. Examples of C₁₋₄ fluoroalkyl include, but are notlimited to, —CF₃, CF₂H, —C₂F₅, and —C₂F₄H.

C₃₋₆ cycloalkyl: the term ‘C₃₋₆ cycloalkyl’ as used herein, pertains toa monovalent moiety obtained by removing a hydrogen atom from a carbonatom of a saturated cyclic core having 3, 4, 5 or 6 atoms in the cycliccore all of which are carbon atoms. Examples of C₃₋₆ cycloalkyl include,but are not limited to, cyclopropyl, cyclohexyl and cyclopentyl.

C₃₋₄ cycloalkyl: the term ‘C₃₋₄ cycloalkyl’ as used herein, pertains toa monovalent moiety obtained by removing a hydrogen atom from a carbonatom of a saturated cyclic core having 3 or 4 atoms in the cyclic coreall of which are carbon atoms. Examples of C₃₋₄ cycloalkyl includecyclopropyl and cyclobutyl.

C₅₋₆ heteroaryl: the term C₅₋₆ heteroaryl as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a ring atomof an aromatic structure having between one and three atoms that are notcarbon forming part of said ring. Wherein, those atoms that are notcarbon can be chosen independently from the list nitrogen, oxygen andsulphur. The group may be substituted by one or more C₁₋₄ alkyl groups.

Examples of C₅₋₆ heteroaryl groups include, but are not limited to,groups derived from:

N₁: pyridine (C₆);

N₁O₁: oxazole (C₅), isoxazole (C₅);

N₂O₁: oxadiazole (furazan) (C₅);

N₂S₁: thiadiazole (C₅)

N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);

N₃: triazole (C₅).

C₅₋₆ heteroaryl methyl: —CH₂—(C₅₋₆ heteroaryl), wherein C₅₋₆ heteroarylis as defined above.

C₄₋₆ heterocyclyl: The term “C₄₋₆ heterocyclyl” as used herein, pertainsto a monovalent moiety obtained by removing a hydrogen atom from a ringatom of a monocyclic heterocyclic compound, which moiety has from 4 to 6ring atoms; of which from 1 to 2 atoms are heteroatoms, chosen fromoxygen or nitrogen.

In this context, the prefixes (e.g. C₄₋₆) denote the number of ringatoms, or range of number of ring atoms, whether carbon atoms orheteroatoms.

Examples of C₄₋₆ heterocyclyl groups include, but are not limited to,those derived from:

N₁: azetidine (C₄), pyrrolidine (tetrahydropyrrole) (C₅), pyrroline(e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrrole or 3H-pyrrole(isopyrrole, isoazole) (C₅), piperidine (C₆), dihydropyridine (C₆),tetrahydropyridine (C₆), azepine (C₇);

N₂: diazetidine (C₄), imidazolidine (C₅), pyrazolidine (diazolidine)(C₅), imidazoline (C₅), pyrazoline (dihydropyrazole) (C₅), piperazine(C₆);

O₁: oxetane (C₄), tetrahydrofuran (C₅); oxane (C₆);

O₂: dioxetane (C₄), dioxolane (C₅); dioxane (C₆);

N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole(C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆),dihydrooxazine (C₆), oxazine (C₆).

Those C₄₋₆ heterocyclyl groups which include a N atom may be substitutedon this atom by an acyl group, and in particular, by —C(═O)Me.

C₄₋₆ heterocyclyl methyl: —CH₂—(C₄₋₆ heterocyclyl), wherein C₄₋₆heterocyclyl is as defined above.

Phenyl: —C₆H₅, wherein the phenyl may itself be optionally substitutedby one or more C₁₋₄ alkyl groups, one or more C₁₋₄ fluoroalkyl groups,one or more C₁₋₄ alkoxy groups, one or more halo substituents and one ormore cyano substituents.

Benzyl: —CH₂-Phenyl, wherein phenyl is as defined above.

Halo: The term “halo” as used herein, refers to a group selected fromfluoro, chloro, bromo and iodo.

Amido: —(C═O)NRR′ wherein R and R′ are independently selected from H,C₁₋₄ alkyl and C₄₋₆ heterocyclyl as defined above, or together form agroup selected from (—CH₂—)_(n) and —(CH₂)_(m)—X—(CH₂)_(p)—, wheren=3-6, m and p=0-4, and X═O or NH. X may also be N—S(═O)₂, S or S(═O)₂.The cyclic amido groups may also be bridged by a further group selectedfrom (—CH₂—)_(n1) and —(CH₂)_(m1)—X—(CH₂)_(p1)—, where n1 is 1-3 and m1and p1 are 1-3. The cyclic amido groups may also be further substitutedby one, two or more hydroxy, oxo, C₁₋₂ alkyl, C₁₋₂ alkyl-C₁₋₂ alkoxy,C₁₋₂ alkyl-hydroxy and C₁₋₂ alkoxy groups or one spiro C₄₋₆ heteroarylor spiro C₄₋₆ cycloalkyl group or be fused to an C₅₋₇ aromatic ring.Examples of amido groups include, but are not limited to, —C(═O)NH₂,—C(═O)NMe₂, —C(═O)NHMe, —C(═O)NHCH(CH₃)₂,

Amidomethyl: —CH₂-amido, where amido is as defined above, Examples ofamidomethyl groups include, but are not limited to, —CH₂—C(═O)NH₂,—CH₂—C(═O)NMe₂, —CH₂—C(═O)NHMe,

Acylamido: —NR(C═O)R′ wherein R and R′ are independently selected fromH, C₁₋₄ alkyl and C₁₋₄ fluoro alkyl as defined above. R′ may also be—(CH₂)_(n)—, where n is 3 or 4. Examples of an acylamido group include,but are not limited to, —N(H)C(═O)CF₃, —N(H)C(═O)Me, and:

Acylamidomethyl: —CH₂-acylamido, where acylamido is as defined above,Examples of acylamidomethyl groups include, but are not limited to—CH₂—N(H)C(═O)Me and —CH₂—N(H)C(═O)CF₃.

C₁₋₄ alkyl ester: —C(═O)OR, wherein R is a C₁₋₄ alkyl group. Examples ofC₁₋₄ alkyl ester groups include, but are not limited to, —C(═O)OCH₃,—C(═O)OCH₂CH₃, and —C(═O)OC(CH₃)₃.

C₁₋₄ alkyl ester methyl: —CH₂—(C₁₋₄ alkyl ester), where C₁₋₄ alkyl esteris as defined above. Examples of C₁₋₄ alkyl ester methyl groups include,but are not limited to, —CH₂—C(═O)OCH₃, —CH₂—C(═O)OCH₂CH₃, and—CH₂—C(═O)OC(CH₃)₃.

C₁₋₄ alkyl carbamoyl: —NHC(═O)OR wherein R is a C₁₋₄ alkyl group asdefined above. Examples of C₁₋₄ alkyl carbamoyl include, but are notlimited to, —N(H)C(═O)OCH₃, —N(H)C(═O)OCH₂CH₃, and —N(H)C(═O)OC(CH₃)₃.

C₁₋₄ alkyl carbamoyl methyl: —CH₂—(C₁₋₄ alkyl carbamoyl), where C₁₋₄alkyl carbamoyl is as defined above. Examples of C₁₋₄ alkyl carbamoylmethyl include, but are not limited to, —CH₂—N(H)C(═O)OCH₃,—CH₂—N(H)C(═O)OCH₂CH₃, and —CH₂—N(H)C(═O)OC(CH₃)₃.

C₁₋₄ alkylacyl: —C(═O)R, wherein R is a C₁₋₄ alkyl group as definedabove. Examples of C₁₋₄ alkylacyl groups include, but are not limitedto, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl) and —C(═O)C(CH₃)₃(t-butyryl).

C₁₋₄ alkylacyl methyl: —CH₂—(C₁₋₄ alkylacyl), where C₁₋₄ alkylacyl is asdefined above. Examples of C₁₋₄ alkylacylmethyl groups include, but arenot limited to, —CH₂—C(═O)CH₃, —CH₂—C(═O)CH₂CH₃, and —CH₂—C(═O)C(CH₃)₃.

Phenylcarbonyl: —O(═O)-phenyl, where phenyl is as defined above.

Carboxy (carboxylic acid): —C(═O)OH

Carboxymethyl: —CH₂—C(═O)OH.

Ether: —OP, wherein P is chosen from one of the following substituents:C₁₋₄ alkyl, benzyl, phenyl, C₁₋₄ fluoroalkyl, C₅₋₆ heteroaryl, —CH₂—C₅₋₆heteroaryl, C₄₋₆ heterocyclyl, and —CH₂—C₄₋₆ heterocyclyl as definedabove. Examples of an ether include, but are not limited to, —OPh, —OBn,—OCF₃, —OCH₂CF₃, —OCH(CH₃)₂, —OCH₂-cyclopropyl,

-   -   where R is methyl, isopropyl, isobutyl;

The term “C₁₋₄ alkyloxy” refers to the group —OP, where P is C₁₋₄ alkyl.

Amino: —NPP′, wherein P and P′ are independently chosen from H, C₁₋₄alkyl, C₄₋₆ heterocyclyl, phenyl and C₅₋₆ heteroaryl as defined above.Examples of an amine include, but are not limited to, —NH₂,—N(H)pyridazinyl,

Aminomethyl: —CH₂-Amino, where amino is as defined above. Examples ofaminomethyl include, but are not limited to, —CH₂—NH₂ and—CH₂—N(H)pyridazinyl.

Sulfonamido: —SO₂NRR′ wherein R and R′ are independently selected fromH, C₁₋₄ alkyl, phenyl and C₅₋₆ heteroaryl as defined above. Examples ofsulfonamido groups include, but are not limited to, —SO₂N(Me)₂ and—SO₂NPhMe.

Sulfonamino: —NHSO₂R wherein R is selected from C₁₋₄ alkyl, phenyl andC₅₋₆ heteroaryl as defined above. Examples of sulfonamino groupsinclude, but are not limited, to —NHSO₂Me and —NHSO₂Ph

Sulfone: —SO₂R, wherein R is selected from C₁₋₄ alkyl and C₁₋₄fluoroalkyl as defined above. Example of sulfone groups includes but isnot limited to SO₂CF₃.

Sulfoxide: —SOR, wherein R is selected from C₁₋₄ alkyl and C₁₋₄fluoroalkyl as defined above. Example of sulfoxide groups includes butis not limited to SOCF₃.

Nitrile: —CN.

Nitrilemethyl: —CH₂—CN

Fused N-heterocyclic ring: where A is phenyl, it may have a C₅₋₆N₁-containing heterocyclic ring fused to it as a substituent group. TheC₅₋₆ N₁-containing heterocyclic ring may in particular be selected from:

Which may be fused in any orientation, and wherein the N ring atom maybe optionally substituted, for example by a C₁₋₄ alkylacyl group.

Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as wellas conventional protected forms. Similarly, a reference to an aminogroup includes the protonated form (—N⁺HR¹R²), a salt or solvate of theamino group, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66,1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g. —COOH may be —COO⁻), then a salt may be formed witha suitable cation. Examples of suitable inorganic cations include, butare not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earthcations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examplesof suitable organic cations include, but are not limited to, ammoniumion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺,NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions arethose derived from: ethylamine, diethylamine, dicyclohexylamine,triethylamine, butylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline,meglumine, and tromethamine, as well as amino acids, such as lysine andarginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic acidand valeric. Examples of suitable polymeric organic anions include, butare not limited to, those derived from the following polymeric acids:tannic acid, carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Isomers

Certain compounds of the invention may exist in one or more particulargeometric, optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and I-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand I or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or I meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

Compounds of the present invention have at least two stereocentres,indicated by * in the formula below:

It may be preferred the compounds have the following stereochemistry:

Alternatively, the compounds may have one of the followingstereochemistries:

Furthermore, the compounds may have the following stereochemistry:

Assignment of the absolute configuration can be determined with X-raycrystallisation studies as described below.

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Examples of isotopes that can be incorporated into compounds of theinvention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, and chlorine, such as, but not limited to ²H(deuterium, D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S,³⁶Cl, and ¹²⁵I. Various isotopically labeled compounds of the presentinvention, for example those into which radioactive isotopes such as 3H,13C, and 14C are incorporated. Such isotopically labelled compounds maybe useful in metabolic studies, reaction kinetic studies, detection orimaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays, or in radioactive treatment ofpatients. Deuterium labelled or substituted therapeutic compounds of theinvention may have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism, and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements. An18F labeled compound may be useful for PET or SPECT studies.Isotopically labeled compounds of this invention and prodrugs thereofcan generally be prepared by carrying out the procedures disclosed inthe schemes or in the examples and preparations described below bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent. Further, substitution with heavierisotopes, particularly deuterium (i.e., 2H or D) may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements or animprovement in therapeutic index. It is understood that deuterium inthis context is regarded as a substituent. The concentration of such aheavier isotope, specifically deuterium, may be defined by an isotopicenrichment factor. In the compounds of this invention any atom notspecifically designated as a particular isotope is meant to representany stable isotope of that atom.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g. fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

Therapeutic Indications

Compounds disclosed herein may provide a therapeutic benefit in a numberof disorders, in particular, in the treatment or prevention of cancersand hemoglobinopathies.

Cancer

Modulators of PRMT5 mediated post-translational arginine methylation ofp53 may regulate a pro-apoptotic p53 response, and may therefore beuseful as therapeutic agents, for example in the treatment of cancer.Such agents may also be useful as therapeutic agents for the treatmentof cancers which exhibit overexpression of PRMT5.

A “cancer” may be any form of cancer. In particular, a cancer cancomprise any one or more of the following: leukemia, acute lymphocyticleukemia (ALL), acute myeloid leukemia (AML), chronic lymphocyticleukemia (CLL), chronic myeloid leukemia (CML), non-Hodgkin's lymphoma,Hodgkin's disease, prostate cancer, lung cancer, melanoma, breastcancer, colon and rectal cancer, colon cancer, squamous cell carcinomaand gastric cancer.

Alternatively, the cancer may comprise adrenocortical cancer, analcancer, bladder cancer, blood cancer, bone cancer, brain tumor, cancerof the female genital system, cancer of the male genital system, centralnervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma,childhood sarcoma, endometrial cancer, endometrial sarcoma, esophagealcancer, eye cancer, gallbladder cancer, gastrointestinal tract cancer,hairy cell leukemia, head and neck cancer, hepatocellular cancer,hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngealcancer, liver cancer, malignant fibrous histiocytoma, malignant thymoma,mesothelioma, multiple myeloma, myeloma, nasal cavity and paranasalsinus cancer, nasopharyngeal cancer, nervous system cancer,neuroblastoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma,ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer,pharyngeal cancer, pituitary tumor, plasma cell neoplasm, primary CNSlymphoma, rectal cancer, respiratory system, retinoblastoma, salivarygland cancer, skin cancer, small intestine cancer, soft tissue sarcoma,stomach cancer, stomach cancer, testicular cancer, thyroid cancer,urinary system cancer, uterine sarcoma, vaginal cancer, vascular system,Waldenstrom's macroglobulinemia and/or Wilms' tumor. Cancers may be of aparticular type. Examples of types of cancer include lymphoma, melanoma,carcinoma (e.g. adenocarcinoma, hepatocellular carcinoma, medullarycarcinoma, papillary carcinoma, squamous cell carcinoma), astrocytoma,glioma, medulloblastoma, myeloma, meningioma, neuroblastoma, sarcoma(e.g. angiosarcoma, chrondrosarcoma, osteosarcoma).

The cancer may be a PRMT5 overexpressing cancer. The cancer may overexpress PRMT5 protein relative to non-cancerous tissue. In some cases,the cancer overproduces PRMT5 mRNA relative to non-cancerous tissue.

Alternatively or additionally, the cancer may be a p53 overexpressingcancer. The cell may overexpress p53 protein relative to non-canceroustissue. It may overproduce p53 mRNA as compared to non-cancerous tissue.In some cases, the level of p53 protein and/or mRNA in the cell is at alevel approximately equivalent to that of a non-cancerous cell.

The agents described herein may be useful in combination with otheranti-cancer therapies. They may act synergistically with chemo- orradiotherapy, and/or with p53 targeted drugs.

An inhibitor of PRMT5 would in all likelihood augment the effects ofdrugs (such as the nutlins) that restore p53. Inhibition of PRMT5,resulting in decreased arginine-methylated p53, may sensitize tumourcells to chemo- and radiotherapy by switching, or at least biasing, thecellular outcome to apoptosis.

Combination Therapies

p53 is activated by DNA damage. PRMT5 is part of the complex of proteinsthat activate and modulate p53 activity in response to DNA damage. It islikely that inhibition of PRMT5, resulting in decreasedarginine-methylated p53, would sensitize tumour cells to chemo- andradiotherapy by switching or at least biasing the cellular outcome toapoptosis. PRMT5 inhibition is likely to synergize well with low dosechemo- or radiotherapy, by stabilizing p53, and biasing the cellularoutcome to apoptosis.

Biasing the p53 response towards apoptosis would in all likelihood be ofbenefit, and an agent that so biases the response would be expected toaugment the effect of a p53 resurrecting drug. Thus, in some cases, aPRMT5 modulator disclosed herein may be administered in conjunction witha radiotherapeutic or chemotherapeutic regime. It may be administered inconjunction with a drug that resurrects cellular p53 activity, forexample, a p53 agonist. The PRMT5 modulator may be administeredsimultaneously or sequentially with radio and/or chemotherapy. Suitablechemotherapeutic agents and radiotherapy protocols will be readilyappreciable to the skilled person. In particular, the compound describedherein may be combined with low dose chemo or radio therapy. Appropriatedosages for “low dose” chemo or radio therapy will be readilyappreciable to the skilled practitioner.

Hemoglobinopathies

The compounds disclosed herein may be useful in the treatment orprevention of conditions that may benefit from the increased expressionof γ-globin genes, for example, due to the release of repressivemethylation of these genes. The compounds disclosed herein may be usefulin the treatment or prevention of hemoglobinopathies. A hemoglobinopathyis a condition associated with the presence of abnormal hemoglobin inthe blood of a subject.

Such conditions include ß-thalassemia and Sickle Cell Disease,α-thalassemia and δ-thalassemia.

Hemoglobinopathies treatable by the compounds disclosed herein may beameliorated by the re-activation of the subjects γ-globin genes (γgenes). In such cases, the subject is not a fetal mammal.

Methods of Treatment

The compounds of the present invention may be used in a method oftherapy. Also provided is a method of treatment, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a compound of the invention. Theterm “therapeutically effective amount” is an amount sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage, is within the responsibility of general practitioners and othermedical doctors.

As described above, the anti cancer treatment defined herein may beapplied as a sole therapy or may involve, in addition to the compound ofthe invention, conventional surgery or radiotherapy or chemotherapy.Such chemotherapy may include one or more of the following categories ofanti-tumour agents:—

(i) other antiproliferative/antineoplastic drugs and combinationsthereof, as used in medical oncology, such as alkylating agents (forexample cisplatin, oxaliplatin, carboplatin, cyclophosphamide, nitrogenmustard, melphalan, chlorambucil, busulphan, temozolamide andnitrosoureas); antimetabolites (for example gemcitabine and antifolatessuch as fluoropyrimidines like 5 fluorouracil and tegafur, raltitrexed,methotrexate, cytosine arabinoside, and hydroxyurea); antitumourantibiotics (for example anthracyclines like adriamycin, bleomycin,doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin); antimitotic agents (for example vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like taxol and docetaxel (Taxotere) and polokinase inhibitors);and topoisomerase inhibitors (for example epipodophyllotoxins likeetoposide and teniposide, amsacrine, topotecan and camptothecin);

(ii) cytostatic agents such as antioestrogens (for example tamoxifen,fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene),antiandrogens (for example bicalutamide, flutamide, nilutamide andcyproterone acetate), LHRH antagonists or LHRH agonists (for examplegoserelin, leuprorelin and buserelin), progestogens (for examplemegestrol acetate), aromatase inhibitors (for example as anastrozole,letrozole, vorazole and exemestane) and inhibitors of 5*-reductase suchas finasteride;

(iii) anti-invasion agents (for example c-Src kinase family inhibitorslike4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-tetrahydropyran-4-yloxyquinazoline(AZD0530; International Patent Application WO 01/94341),N-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-ylamino}thiazole-5-carboxamide(dasatinib, BMS-354825; J. Med. Chem., 2004, 47, 6658-6661 and and4-((2,4-dichloro-5-methoxyphenyl)amino)-6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinoline-3-carbonitrile(bosutinib, SKI-606; Cancer research (2003), 63(2), 375-81), andmetalloproteinase inhibitors like marimastat, inhibitors of urokinaseplasminogen activator receptor function or antibodies to Heparanase);

(iv) inhibitors of growth factor function: for example such inhibitorsinclude growth factor antibodies and growth factor receptor antibodies(for example the anti erbB2 antibody trastuzumab [HerceptinT], theanti-EGFR antibody panitumumab, the anti erbB1 antibody cetuximab[Erbitux, C225] and any growth factor or growth factor receptorantibodies disclosed by Stern et al. Critical reviews inoncology/haematology, 2005, Vol. 54, pp 11-29); such inhibitors alsoinclude tyrosine kinase inhibitors, for example inhibitors of theepidermal growth factor family (for example EGFR family tyrosine kinaseinhibitors such asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, ZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI 774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine(CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib,inhibitors of the hepatocyte growth factor family, inhibitors of theplatelet-derived growth factor family such as imatinib, inhibitors ofserine/threonine kinases (for example Ras/Raf signalling inhibitors suchas farnesyl transferase inhibitors, for example sorafenib (BAY43-9006)), inhibitors of cell signalling through MEK and/or AKT kinases,inhibitors of the hepatocyte growth factor family, c-kit inhibitors, ablkinase inhibitors, IGF receptor (insulin-like growth factor) kinaseinhibitors; aurora kinase inhibitors (for example AZD1152, PH739358,VX-680, MLN8054, R763, MP235, MP529, VX-528 AND AX39459) and cyclindependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors;

(v) antiangiogenic and antilymphangiogenic agents such as those whichinhibit the effects of vascular endothelial growth factor, [for examplethe anti vascular endothelial cell growth factor A (VEGFA) antibodybevacizumab (AvastinT), the anti vascular endothelial cell growth factorA (VEGFA) antibody ranibizumab, the anti-VEGF aptamer pegaptanib, theanti vascular endothelial growth factor receptor 3 (VEGFR3) antibodyIMC-3C5, the anti vascular endothelial cell growth factor C (VEGFC)antibody VGX-100, the anti vascular endothelial cell growth factor D(VEGFD) antibody VGX-200, the soluble form of the vascular endothelialgrowth factor receptor 3 (VEGFR3) VGX-300 and VEGF receptor tyrosinekinase inhibitors such as4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(vandetanib; ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(cediranib; AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787;WO 98/35985), pazopanib (GW786034), axitinib (AG013736), sorafenib andsunitinib (SU11248; WO 01/60814), compounds such as those disclosed inInternational Patent Applications WO97/22596, WO 97/30035, WO 97/32856and WO 98/13354 and compounds that work by other mechanisms (for examplelinomide, inhibitors of integrin avb3 function and angiostatin)];

(vi) vascular damaging agents such as Combretastatin A4 and compoundsdisclosed in International Patent Applications WO 99/02166, WO 00/40529,WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;

(vii) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense;

(viii) gene therapy approaches, including for example approaches toreplace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2,GDEPT (gene directed enzyme pro drug therapy) approaches such as thoseusing cytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi drug resistance gene therapy; and

(ix) immunotherapy approaches, including for example ex vivo and in vivoapproaches to increase the immunogenicity of patient tumour cells, suchas transfection with cytokines such as interleukin 2, interleukin 4 orgranulocyte macrophage colony stimulating factor, approaches to decreaseT cell anergy, approaches using transfected immune cells such ascytokine transfected dendritic cells, approaches using cytokinetransfected tumour cell lines and approaches using anti idiotypicantibodies

Administration

The active compound or pharmaceutical composition comprising the activecompound may be administered to a subject by any convenient route ofadministration, whether systemically/peripherally or at the site ofdesired action, including but not limited to, oral (e.g. by ingestion);topical (including e.g. transdermal, intranasal, ocular, buccal, andsublingual); pulmonary (e.g. by inhalation or insufflation therapyusing, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal;parenteral, for example, by injection, including subcutaneous,intradermal, intramuscular, intravenous, intraarterial, intracardiac,intrathecal, intraspinal, intracapsular, subcapsular, intraorbital,intraperitoneal, intratracheal, subcuticular, intraarticular,subarachnoid, intravitreal and intrasternal; by implant of a depot, forexample, subcutaneously, intravitreal or intramuscularly. The subjectmay be a eukaryote, an animal, a vertebrate animal, a mammal, a rodent(e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse),canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), aprimate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset,baboon), an ape (e.g. gorilla, chimpanzee, orangutan, gibbon), or ahuman.

Formulations

While it is possible for the active compound to be administered alone,it is preferable to present it as a pharmaceutical composition (e.g.formulation) comprising at least one active compound, as defined above,together with one or more pharmaceutically acceptable carriers,adjuvants, excipients, diluents, fillers, buffers, stabilisers,preservatives, lubricants, or other materials well known to thoseskilled in the art and optionally other therapeutic or prophylacticagents.

Thus, the present invention further provides pharmaceuticalcompositions, as defined above, and methods of making a pharmaceuticalcomposition comprising admixing at least one active compound, as definedabove, together with one or more pharmaceutically acceptable carriers,excipients, buffers, adjuvants, stabilisers, or other materials, asdescribed herein.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of a subject (e.g. human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation.

Suitable carriers, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Suchmethods include the step of bringing into association the activecompound with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

Formulations may be in the form of liquids, solutions, suspensions,emulsions, elixirs, syrups, tablets, losenges, granules, powders,capsules, cachets, pills, ampoules, suppositories, pessaries, ointments,gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses,electuaries, or aerosols.

Formulations suitable for oral administration (e.g. by ingestion) may bepresented as discrete units such as capsules, cachets or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or suspension in an aqueous or non-aqueousliquid; or as an oil-in-water liquid emulsion or a water-in-oil liquidemulsion; as a bolus; as an electuary; or as a paste.

A tablet may be made by conventional means, e.g., compression ormoulding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing in a suitable machine the activecompound in a free-flowing form such as a powder or granules, optionallymixed with one or more binders (e.g. povidone, gelatin, acacia,sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers ordiluents (e.g. lactose, microcrystalline cellulose, calcium hydrogenphosphate); lubricants (e.g. magnesium stearate, talc, silica);disintegrants (e.g. sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose); surface-active ordispersing or wetting agents (e.g. sodium lauryl sulfate); andpreservatives (e.g. methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,sorbic acid). Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activecompound therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile. Tablets mayoptionally be provided with an enteric coating, to provide release inparts of the gut other than the stomach.

Formulations suitable for topical administration (e.g. transdermal,intranasal, ocular, buccal, and sublingual) may be formulated as anointment, cream, suspension, lotion, powder, solution, past, gel, spray,aerosol, or oil. Alternatively, a formulation may comprise a patch or adressing such as a bandage or adhesive plaster impregnated with activecompounds and optionally one or more excipients or diluents.

Formulations suitable for topical administration in the mouth includelosenges comprising the active compound in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activecompound in an inert basis such as gelatin and glycerin, or sucrose andacacia; and mouthwashes comprising the active compound in a suitableliquid carrier.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active compound is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the active compound.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of about 20 to about 500 microns which is administered in themanner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid for administrationas, for example, nasal spray, nasal drops, or by aerosol administrationby nebuliser, include aqueous or oily solutions of the active compound.

Formulations suitable for administration by inhalation include thosepresented as an aerosol spray from a pressurised pack, with the use of asuitable propellant, such as dichlorodifluoromethane,trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, orother suitable gases.

Formulations suitable for topical administration via the skin includeointments, creams, and emulsions. When formulated in an ointment, theactive compound may optionally be employed with either a paraffinic or awater-miscible ointment base. Alternatively, the active compounds may beformulated in a cream with an oil-in-water cream base. If desired, theaqueous phase of the cream base may include, for example, at least about30% w/w of a polyhydric alcohol, i.e., an alcohol having two or morehydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol,sorbitol, glycerol and polyethylene glycol and mixtures thereof. Thetopical formulations may desirably include a compound which enhancesabsorption or penetration of the active compound through the skin orother affected areas. Examples of such dermal penetration enhancersinclude dimethylsulfoxide and related analogues.

When formulated as a topical emulsion, the oily phase may optionallycomprise merely an emulsifier (otherwise known as an emulgent), or itmay comprises a mixture of at least one emulsifier with a fat or an oilor with both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabiliser. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabiliser(s) make up theso-called emulsifying wax, and the wax together with the oil and/or fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodiumlauryl sulphate. The choice of suitable oils or fats for the formulationis based on achieving the desired cosmetic properties, since thesolubility of the active compound in most oils likely to be used inpharmaceutical emulsion formulations may be very low. Thus the creamshould preferably be a non-greasy, non-staining and washable productwith suitable consistency to avoid leakage from tubes or othercontainers. Straight or branched chain, mono- or dibasic alkyl esterssuch as di-isoadipate, isocetyl stearate, propylene glycol diester ofcoconut fatty acids, isopropyl myristate, decyl oleate, isopropylpalmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branchedchain esters known as Crodamol CAP may be used, the last three beingpreferred esters. These may be used alone or in combination depending onthe properties required.

Alternatively, high melting point lipids such as white soft paraffinand/or liquid paraffin or other mineral oils can be used.

Formulations suitable for rectal administration may be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active compound, such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration (e.g. by injection,including cutaneous, subcutaneous, intramuscular, intravenous andintradermal), include aqueous and non-aqueous isotonic, pyrogen-free,sterile injection solutions which may contain anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. Examples of suitable isotonic vehicles for use insuch formulations include Sodium Chloride Injection, Ringer's Solution,or Lactated Ringer's Injection. Typically, the concentration of theactive compound in the solution is from about 1 ng/mL to about 10 μg/mL,for example from about 10 ng/ml to about 1 μg/mL. The formulations maybe presented in unit-dose or multi-dose sealed containers, for example,ampoules and vials, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets. Formulations may be in the form ofliposomes or other microparticulate systems which are designed to targetthe active compound to blood components or one or more organs.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the compound, and compositions comprising the compound, canvary from patient to patient. Determining the optimal dosage willgenerally involve the balancing of the level of therapeutic benefitagainst any risk or deleterious side effects. The selected dosage levelwill depend on a variety of factors including, but not limited to, theactivity of the particular compound, the route of administration, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds, and/or materials usedin combination, the severity of the condition, and the species, sex,age, weight, condition, general health, and prior medical history of thepatient. The amount of compound and route of administration willultimately be at the discretion of the physician, veterinarian, orclinician, although generally the dosage will be selected to achievelocal concentrations at the site of action which achieve the desiredeffect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the active compound is in the range ofabout 100 ng to about 25 mg (more typically about 1 μg to about 10 mg)per kilogram body weight of the subject per day. Where the activecompound is a salt, an ester, an amide, a prodrug, or the like, theamount administered is calculated on the basis of the parent compoundand so the actual weight to be used is increased proportionately.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 100 mg, 3 timesdaily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 150 mg, 2 timesdaily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 200 mg, 2 timesdaily.

However in one embodiment, the active compound is administered to ahuman patient according to the following dosage regime: about 50 orabout 75 mg, 3 or 4 times daily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 100 or about 125mg, 2 times daily.

Treatment

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, regression of the condition,amelioration of the condition, and cure of the condition. Treatment as aprophylactic measure (i.e., prophylaxis, prevention) is also included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of an active compound, or a material, composition or dosagefrom comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio, when administered in accordance with a desiredtreatment regimen.

Similarly, the term “prophylactically-effective amount,” as used herein,pertains to that amount of an active compound, or a material,composition or dosage from comprising an active compound, which iseffective for producing some desired prophylactic effect, commensuratewith a reasonable benefit/risk ratio, when administered in accordancewith a desired treatment regimen.

The Subject/Patient

The subject/patient may be an animal, mammal, a placental mammal, amarsupial (e.g., kangaroo, wombat), a monotreme (e.g., duckbilledplatypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse),murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., abird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., ahorse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., acow), a primate, simian (e.g., a monkey or ape), a monkey (e.g.,marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang,gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development,for example, a foetus. In one preferred embodiment, the subject/patientis a human.

General Synthesis Methods

The compounds of the invention can be prepared employing the followinggeneral methods and using procedures described in detail in theexamples. The reaction conditions referred to are illustrative andnon-limiting, for example one skilled in the art may use a diverse rangeof synthetic methods to synthesis the desired compounds such as but notlimited to methods described in literature (for example but not limitedto March's Advanced Organic Chemistry: R^(2a)ctions, Mechanisms, andStructure, 7th Edition or Larock's Comprehensive OrganicTransformations: Comprehensive Organic Transformations: A Guide toFunctional Group Preparations).

Compounds of formula I, as described above, can be prepared by syntheticstrategies outlined below, wherein the definitions above apply.

General Synthesis 1a

Scheme 1A illustrates the synthesis of compounds with the structure G9where R^(2a) and R^(2c)═H. A coupling of a carbonyl compound ofstructure G1 with an organometallic compound of structure G2 to give acompound with structure G3 will be apparent to those skilled in the art.The group represented by (M) includes but is not limited to Mg, In, Znand the group represented by (X) may be a halide where (Y) may be thenumber 1-3. Suitable protected amino groups represented by (PG) includebut are not limited to phthalimide; and methods for the removal of saidprotecting groups will be known to those skilled in the art (for exampleGreene's Protective Groups in Organic Synthesis, 4th Edition). Synthesisof compounds with structure G5 is performed by reacting alkyne G3 withcompounds of structure G4 in the presence of a transition metal catalystor combination of transition metal catalysts such as but not limited tobis(triphenylphosphine)nickel(II) chloride/Zn.

After removal of the protecting group, for R⁵=Me, methods to synthesisG6 will require an additional methylation reaction apparent to thoseskilled in the art, but include for example, a reductive amination withformaldehyde and a reducing agent or combination of reducing agents suchas but not limited to formic acid, formic acid derivatives, NaBH₄,NaCNBH₄, NaBH(OAc)₃ or NaBH₃CN—Ti(Oi-Pr)₄. Methods to synthesise amidesG8 will be apparent to those skilled in the art, but include for examplethe use of reagents such as HATU, HBTU, T3P and EDCI/HOBt, and the useof activated forms of the carboxylic acid G7 such as the correspondingacyl halide, carbamate or N-hydroxysuccinimide ester. Transformation ofisoquinolines of structure G8 to give tetrahydroisoquinolines ofstructure G9 will be apparent to those skilled in the art and suchmethods include but are not limited to reduction in the presence of atransition metal catalyst. For R^(2e)=Me, methods to synthesis G9 willrequire an additional methylation reaction apparent to those skilled inthe art, but include for example, a reductive amination withformaldehyde and a reducing agent or combination of reducing agents suchas but not limited to formic acid, formic acid derivatives, NaBH₄,NaCNBH₄, NaBH(OAc)₃ or NaBH₃CN—Ti(Oi-Pr)₄.

Alternatively, for the synthesis of compounds with structure G12 whereR^(2b), R^(2c), R^(2d)═H, a coupling of an alkyne and an aryl halidewill be apparent to those skilled in the art and such methods include acoupling in the presence of a transition metal catalyst or catalystcombination such as but not limited to PdCl₂(PPh₃)₂/CuI andPd(OAc)₂/PPh₃. This may be followed by cyclisation either in situ or asa separate step to give isoquinoline of structure G11. The syntheticsteps to give compounds with general formula G12 will be similar tothose used in Scheme 1A.

General Synthesis 1b

Scheme 1b illustrates the synthesis of intermediate compounds X7/G19,beginning with a Pictet-Spengler reaction between a β-arylethylamine X1and an aldehyde (or ketone where R^(2c) and R^(2d) is not ═H) X2 in thepresence or absence of a suitable acid, such as but not limited to HCl,TFA, H₂SO₄ or BF₃.OEt₂ to furnish tetrahydroisoquinoline X3. Protectionof the amine using a suitable protecting group will be apparent to thoseskilled in the art (for example Greene's Protective Groups in OrganicSynthesis, 4th Edition) and gives intermediate X4. Reduction of the acidgroup to the primary alcohol X5 will be apparent to those skilled in theart and include but are not limited to the use of reagents such asNaBH₄/CDI, LiBH₄, LiAlH₄, and borane complexes. Oxidation of the alcoholto the corresponding aldehyde X7 will be apparent to those skilled inthe art and include but are not limited to the use of reagents such asDess-Martin periodinane, IBX or dimethylsulfoxide combined with asuitable electrophilic reagent such as but not limited topyridine-sulfur trioxide. Alternatively, methods to form the Weinrebamide X6 will be apparent to those skilled in the art, but include forexample the use of reagents such as but not limited toN,O-dimethylhydroxylamine and HATU, HBTU, T3P and EDCI/HOBt, or the useof activated forms of the carboxylic acid such as the corresponding acylhalide, mixed anhydride or N-hydroxysuccinimide ester. Reduction of theamide to the aldehyde (R⁴═H) will be apparent to those skilled in theart and include but are not limited to reagents such as LiAlH₄. ForR⁴=Me, treatment of the intermediate amide X6 with a suitableorganometallic reagent such as but not limited to MeLi or MeMgBrfurnishes X7/G19.

General Synthesis 2

Where R⁷ represents the fused ring group.

Scheme 2A illustrates the synthesis of compounds G18 from aldehyde G13(or ketone where R⁴=Me). Conversion of a carbonyl to an alkene will beapparent to those skilled in the art but methods include but are notlimited to a Wittig reaction with [Ph₃PMe]⁺Br⁻ in the presence of a basesuch as KHMDS. The alkene G14 can be epoxidised with reagents such asm-CPBA and then reacted with an amine to give intermediate G16.Alternatively, an aminohydroxylation can be performed by methods such asbut not limited to reaction with (PG)NHOTs in the presence of potassiumosmate dihydrate. Removal of the protecting group will be apparent tothose skilled in the art (for example Greene's Protective Groups inOrganic Synthesis, 4th Edition) and gives intermediate G17. Amide bondformation to give compounds G18 can be performed by methods previouslydescribed (General synthesis 1).

General Synthesis 3

Where R⁷ represents the fused ring group.

Scheme 3A illustrates the synthesis of compounds G22 beginning with aHenry reaction between an aldehyde G19 (or ketone where R⁴=Me) andnitromethane in the presence or absence of a suitable base, such as butnot limited to DBU, a KF, TBAF or sodium hydroxide, in the presence orabsence of a chiral or achiral transition metal compound for example butnot limited to complexes of copper, cobalt or zinc to furnish anitro-alcohol G20. Reduction of the nitro group to the primary amine G21will be apparent to those skilled in the art and include but are notlimited to using reducing conditions such as a transition metal (Fe, In,Zn) in the presence of HCl, hydrogenation in the presence of atransition metal or transition metal catalyst. Amide bond formation togive compounds G22 can be performed by methods previously described(General synthesis 1). The method can also be carried out withnitroethane and other nitroalkanes, as appropriate.

General Synthesis 4

Scheme 4A illustrates the addition of an amine (HNR⁸R⁹), as asubstituent which is a part of A. This can be achieved by coupling arelevant carboxylic acid to a primary amine or a secondary amine,NHR⁸R⁹. Methods to form such amides will be apparent to those skilled inthe art, but include for example the use of reagents such as HATU, HBTU,T3P and EDCI/HOBt, and the use of activated forms of the carboxylic acidsuch as the corresponding acyl halide, mixed anhydride orN-hydroxysuccinimide ester. The group denoted by (X) may be but notlimited to halogen, tosylate or other suitable group. Conversion of (X)in G22 into an ester in G23 will be apparent to those skilled in theart, but include for example a carbonylation reaction which can beachieved by the use of carbon monoxide in the presence of an transitionmetal catalyst such as but not limited to PdCl₂dppf.DCM; and analcoholic solvent such as but not limited to methanol, ethanol,isopropanol or tert-butyl alcohol. Formation of the carboxylic acid canbe achieved by for example hydrolysis with a base such as an alkalimetal hydroxide or an acid for example aqueous hydrochloric acid to formG24. The amide formation to form G25 can be achieved by the methodsoutline in Scheme 1A.

Alternatively, for the synthesis of ester G24 the order of steps can bereversed as described in Scheme 4B.

Alternatively for the synthesis of amide G25 the steps may be reorderedsuch that the formation of the R⁸R⁹N amide on the A substituent occursafter the coupling of A to the primary amine G21. This may be achievedby coupling a suitable amine with an intermediate where A bears asuitable functional group for coupling, for example but not limited to acarboxylic acid or alkali metal carboxylate salt, as shown in Scheme 4C.

General Synthesis 5

Scheme 5A illustrates the addition of an R¹¹ group, as a substituentwhich is part of A. This can be achieved using any suitable couplingreaction known to the person skilled in the art, for example by Suzukicoupling. The groups denoted by R¹¹X and B¹ are chosen to be suitablefor the coupling reaction employed. For example, in the case of a Suzukicoupling reaction (X) may be a halogen, tosylate or other suitable groupand B¹ represents a suitable boron compound including, but not limitedto, a boronic acid or boronic ester.

Examples of B¹ that can be used in the Suzuki coupling include, but arenot limited to, those shown below.

The types of R¹¹X compounds that can be used in the Suzuki couplinginclude, but are not limited to, those shown in Table 1.

TABLE 1

In addition to scheme 5A, the position of the (X) and (B¹) can bereversed as shown below in scheme 2B, to give the same final compoundG27. Similarly to Scheme 2A, the groups denoted by R¹¹B¹ and (X) arechosen to be suitable for the coupling reaction employed. For example,in the case of a Suzuki coupling reaction (X) may be a halogen, tosylateor other suitable group and R¹¹B¹ represents a suitable boron compoundincluding, but not limited to, a boronic acid or boronic ester.

The types of R¹¹B¹ compounds that can be used in the Suzuki couplinginclude, but are not limited to, those shown in Table 2.

TABLE 2

A variety of coupling reactions may be used to introduce the R¹¹ groupother than Suzuki coupling, such as for example transition metalcatalysed coupling reactions of for example tin (Stille type reaction)and zinc (Negishi type reaction) compounds. Substitution of the halogenby suitable nucleophiles in the presence or absence of other reagentssuch as for example transition metal compounds is also suitable.

Coupling reactions can also be used to prepare the carboxylic acids usedin Scheme 1A for the amide formations, scheme 5C. In starting materialG30 and G32, A as described herein, consists of -A²X and -A²B¹respectively. In the product G33, A as described herein, consists of-A²R¹¹. The groups denoted by (X) and B¹ are chosen to be suitable forthe coupling reaction employed. For example, in the case of a Suzukicoupling reaction (X) may be a halogen, tosylate or other suitable groupand B¹ represents a suitable boron compound including, but not limitedto, a boronic acid or boronic ester.

In G30 and G32 R¹² can be a H or a carbon group for example but notlimited to Me, Et, Pr, iPr, Bu, t-Bu. In these instances where R¹² iscarbon group it may be necessary to form the carboxylic acid before usein the amide coupling (Scheme 1A), generally this can be achieved by forexample hydrolysis with a base such as an alkali metal hydroxide or anacid for example aqueous hydrochloric acid to form G33. The same methodfor converting an ester to a carboxylic acid is used in other generalschemes.

General Synthesis 6

Scheme 6A illustrates the addition of an R¹³ group, as a substituentwhich is part of A. This can be achieved using any suitable couplingreaction known to the person skilled in the art, for example, by an SnArdisplacement or Buchwald coupling. The group denoted by (X) may be butnot limited to halogen and is chosen to be suitable for the couplingreaction employed.

In G34 and G35 R¹⁴ can be a H or a carbon group for example but notlimited to Me, Et, Pr, iPr, Bu, t-Bu. In these instances it may benecessary to form the carboxylic acid before use in an amide coupling(Scheme 1A), generally this can be achieved by, for example, hydrolysiswith a base such as an alkali metal hydroxide or an acid, for example,aqueous hydrochloric acid to form G36. The same method for converting anester to a carboxylic acid is used in other general schemes.

This method may also be extended to the addition of secondary amines.

Alternatively, to synthesise ether linked compounds, a similar strategycan be employed as shown in Scheme 6B. This can be achieved using anysuitable coupling reaction known to a person skilled in the art, forexample, by an S_(N)Ar displacement or an Ullman-type coupling to givecompounds with structure G37. Upon hydrolysis using methods previouslydescribed, compounds with structure G38 may be obtained and used in anamide bond formation as shown in scheme 1A.

Both the above couplings may also be reversed, such that the group addedis R¹³—X.

Further Embodiments

n

In some embodiments, n is 1. In some embodiments, n is 2.

p

In some embodiments, p is 0. In some embodiments, p is 1.

R^(1a), R^(1b), R^(1c) and R^(1d)

In some embodiments, R^(1a), R^(1b), R^(1c) and R^(1d) are all H. Insome embodiments, one of R^(1a), R^(1b), R^(1c) and R^(1d) is halo, C₁₋₄alkoxy, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄ cycloalkyl, NH—C₁₋₄ alkyl orcyano. In some of there embodiments, R^(1b) or R^(1c) is halo, C₁₋₄alkoxy, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄ cycloalkyl, NH—C₁₋₄ alkyl orcyano, and in further of these embodiments, R^(1c) is halo, C₁₋₄ alkoxy,C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄ cycloalkyl, NH—C₁₋₄ alkyl or cyano.Where one of R^(1a), R^(1b), R^(1c) and R^(1d) is halo, C₁₋₄ alkoxy,C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄ cycloalkyl, NH—C₁₋₄ alkyl or cyano,it may be that the group is chloro, methoxy, methyl, ethyl, NHMe orcyano. In some embodiments, the group is methoxy.

In further embodiments, R^(1c) is selected from methoxy, ethoxy, ethyl,CHF₂, CF₃ and cyano. In some of these embodiments, R^(1c) is selectedfrom methoxy, ethyl, CHF₂, CF₃ and cyano.

R^(2a), R^(2b), R^(2c) and R^(2d)

R^(2a), R^(2b), R^(2c) and R^(2d) are independently selected from H, F,CH₂OH and Me. In some of these embodiments, R^(2a), R^(2b), R^(2c) andR^(2d) are independently selected from H, Me and CH₂OH. In further ofthese embodiments, R^(2a), R^(2b), R^(2c) and R^(2d) are independentlyselected from H and Me.

In some embodiments R^(2a), R^(2b), R^(2c) and R^(2d) are all H.

In some embodiments R^(2a), R^(2b), R^(2c) and R^(2d) are comprised ofthree H and one Me or CH₂OH group. It may be preferred in theseembodiments that R^(2a) is Me and R^(2b), R^(2c) and R^(2d) are H. Itmay be preferred in these embodiments that R^(2c) is Me or CH₂OH andR^(2a), R^(2b) and R^(2d) are H.

In some embodiments R^(2a), R^(2b), R^(2c) and R^(2d) are comprised oftwo H and two Me groups. It may be preferred in these embodiments thatR^(2a) and R^(2c) are Me and R^(2b) and R^(2d) are H. It may bepreferred in these embodiments that R^(2a) and R^(2b) are Me and R^(2c)and R^(2d) are H. It may also be preferred in these embodiments thatR^(2c) and R^(2d) are Me and R^(2a) and R^(2b) are H.

In some embodiments, R^(2c) and R^(2d) are independently selected from Hand F.

R^(2e)

In some embodiments, R^(2e) is H. In other embodiments, R^(2e) is Me.

R³

R^(3a) and R^(3b) are independently selected from H and Me. In someembodiments R^(3a) is H and R^(3b) is Me. In some embodiments R^(3a) andR^(3b) are both H. In some embodiments R^(3a) and R^(3b) are both Me.

R⁴

In some embodiments R⁴ is H. In some embodiments R⁴ is Me.

R⁵

In some embodiments R⁵ is H. In some embodiments R⁵ is Me.

R⁶

R^(6a) and R^(6b) are independently selected from H and Me. In someembodiments R^(6a) is H and R^(6b) is Me. In some embodiments R^(6a) andR^(6b) are both H. In some embodiments R^(6a) and R^(6b) are both Me.

Stereoisomers

The carbon to which R⁴ is attached is a chiral centre.

In some embodiments, the compound is a mixture of stereoisomers at thiscentre. In some embodiments, the compound is a single stereoisomer. Insome of these embodiments, the compound is the (R)-stereoisomer. Inothers of these embodiments, the compound is the (S)-stereoisomer.

The compound may also include further chiral centres. The carbon in theTHIQ moiety is chiral. In some embodiments, the compound is a mixture ofstereoisomers at this centre. In other embodiments, the compound is asingle stereoisomer at this centre. In some of these embodiments, thecompound is the (R)-stereoisomer at this centre. In others of theseembodiments, the compound is the (S)-stereoisomer at this centre.

Thus the compound may be a single diastereomer or a mixture ofdiastereomers.

It may be preferred the compounds have the following stereochemistry:

Alternatively, the compounds may have one of the followingstereochemistries:

R¹-R⁵, n & p

In some embodiments, R^(1a), R^(1b), R^(1d), R^(2a), R^(2b), R^(2c),R^(2d), R⁴, R⁵, R^(6a) and R^(6b) are all H, n is 1 and p is 0, and thusthe compound of formula I is of formula Ia:

wherein R^(1c) is H or C₁₋₄ alkoxy, C₂₋₄ alkyl, NH—C₁₋₄ alkyl or cyano(e.g. methoxy) and R^(2e) is H or methyl, and one of R^(1c) and R^(2e)is not H.

In some embodiments, R^(1a), R^(1b), R^(1d), R^(2a), R^(2b), R^(2c),R^(2d), R^(3a), R^(3b), R⁴, R⁵, R^(6a) and R^(6b) are all H, n is 1 andp is 1, and thus the compound of formula I is of formula Ib:

wherein R^(1c) is H or C₁₋₄ alkoxy, C₂₋₄ alkyl, NH—C₁₋₄ alkyl or cyano(e.g. methoxy) and R^(2e) is H or methyl, and one of R^(1c) and R^(2e)is not H.

In some embodiments, R^(1a), R^(1b), R^(1d), R^(2a), R^(2b), R^(2c),R^(2d), R⁴, R⁵, R^(6a) are all H, n is 1 and p is 0, and R^(6b) is Meand thus the compound of formula I is of formula Ic:

wherein R^(1c) is H or C₁₋₄ alkoxy, C₂₋₄ alkyl, NH—C₁₋₄ alkyl or cyano(e.g. methoxy) and R^(2e) is H or methyl, and one of R^(1c) and R^(2e)is not H.

A

Optional Substituents

When the optional substituent on A is C₁₋₄ alkyl, it may be preferablyselected from methyl, ethyl, i-Pr, t-Bu.

When the optional substituent on A is C₁₋₄ fluoroalkyl, it maypreferably be selected from —CF₃ and —CF₂H.

When the optional substituent on A is C₅₋₆ heteroaryl, it may besubstituted by one or more C₁₋₄ alkyl groups. These groups maypreferably be on one or more of the nitrogen ring atoms (if present).These groups may also preferably be methyl.

Further, when the optional substituent on A is C₅₋₆ heteroaryl, it maybe substituted by a C₃₋₄ alkylene group which is bound to the ring intwo places to form a fused ring structure.

When the optional substituent on A is C₅₋₆ heteroaryl, it may preferablybe selected from pyridizinyl, pyrimidinyl, pyridinyl, pyrazolyl,pyrazinyl, oxadiazolyl, isoxazolyl, triazolyl, imidazolyl,benzimidazolyl and thiadiazolyl.

When the optional substituent on A is C₅₋₆ heteroaryl methyl, it maypreferably be selected from —CH₂-imidazolyl and —CH₂-triazolyl.

When the optional substituent on A is C₅₋₆ heterocyclyl, it maypreferably be morpholino.

When the optional substituent on A is C₅₋₆ heterocyclyl methyl, it maypreferably be selected from —CH₂-morpholino and —CH₂-piperazinyl.

When the optional substituent on A is phenyl, it may be substituted byone or more C₁₋₄ alkyl groups. These groups may preferably be methyl.

When the optional substituent on A is phenyl, it may be substituted byone or more C₁₋₄ fluoroalkyl groups. These groups may preferably betrifluoromethyl.

When the optional substituent on A is phenyl, it may be substituted byone or more C₁₋₄ alkoxy groups. These groups may preferably be methoxy.

When the optional substituent on A is phenyl, it may be substituted byone or more halo substituents. These groups may preferably be fluoro orchloro, more preferably fluoro.

When the optional substituent on A is phenyl, it may be substituted byone or more cyano groups. It may be preferred that there is a singlecyano substituent.

When the optional substituent on A is halo, it may preferably beselected from F, Cl and Br.

When the optional substituent on A is amido, the amido substituentgroups R and R′ may preferably form a ring, which ring may also bebridged or substituted. If the amido group is not cyclic, it maypreferably be selected from —C(═O)NH₂, —C(═O)NMeH, —C(═O)NMe₂ and—C(═O)N^(i)PrH. If the amido group is cyclic, it may preferably beselected from —C(═O)-piperidinyl, —C(═O)-hydroxypiperidinyl,—C(═O)-methoxypiperidinyl, —C(═O)-pyrrolidinyl, —C(═O)-morpholino,—C(═O)-methylmorpholino, —C(═O)-dimethylmorpholino and—C(═O)-azetidinyl. Further cyclic amido groups include:

When the optional substituent on A is amidomethyl, the amido substituentgroups R and R′ may preferably form a ring, which ring may also bebridged or substituted. If the amido group is not cyclic, theamidomethyl group may preferably be selected from —CH₂C(═O)NH₂,—CH₂C(═O)NMeH and —CH₂C(═O)N^(i)PrH. If the amido group is cyclic, theamidomethyl group may preferably be selected from—CH₂C(═O)-pyrrolidinyl-CH₂C(═O)-morpholino, —C(═O)-hydroxypiperidinyl,—C(═O)-methoxypiperidinyl, —C(═O)-methylmorpholino andCH₂C(═O)-azetidinyl. Further cyclic amidomethyl groups include:

When the optional substituent on A is acylamido, it may preferably beγ-lactam.

When the optional substituent on A is acylamidomethyl, it may preferablybe selected from —CH₂NHC(═O)Me, —CH₂NHC(═O)CF₃.

When the optional substituent on A is C₁₋₄ alkyl ester, it maypreferably be —C(═O)—OMe.

When the optional substituent on A is C₁₋₄ alkyl ester methyl, it maypreferably be —CH₂—C(═O)—OMe.

When the optional substituent on A is C₁₋₄ alkyl carbamoyl methyl, itmay preferably be —CH₂NHC(═O)OMe.

When the optional substituent on A is C₁₋₄ alkylacyl, it may preferablybe selected from —C(═O)Me and —C(═O)Et.

When the optional substituent on A is C₁₋₄ alkylacylmethyl, it maypreferably be —CH₂C(═O)Me.

When the optional substituent on A is phenylcarbonyl, it may preferablybe —C(═O)-Ph.

When the optional substituent on A is ether, it may preferably beselected from methoxy, ethoxy, —OBn, —OPh, —OCF₃, —OCF₂H, —O—(C₆H₄)—CN,—O-oxanyl, —OCH₂pyridinyl, —OCH₂₋oxadiazolyl, —OCH₂-isoxazole,

When the optional substituent on A is amino, the amino substituent maybe a C₅₋₆ heteroaryl group, in which case the amino group may preferablybe selected from —NH-pyrazinyl, —NH-pyrimidine. In other embodiments,the amino substituent may be a C₄₋₆ heterocyclyl group, such asoptionally N-substituted azetidinyl, optionally N-substitutedpiperidinyl and oxetanyl. A further amino group may be:

When the optional substituent on A is aminomethyl, it may preferably be—CH₂NH₂. Alternatively, the amino substituent may be as defined above.

When the optional substituent on A is sulfonamido it may preferably beselected from —SO₂NMePh, —SO₂NMe₂, and —SO₂NHEt.

When the optional substituent on A is sulfonamino, it may preferably beselected from —NHSO₂Ph and —NHSO₂Me.

When the optional substituent on A is sulfone, it may preferably be—SO₂CF₃.

Optionally Substituted Phenyl

In some embodiments A may be an optionally substituted phenyl.

In some of these embodiments, A is unsubstituted phenyl.

In some of these embodiments, the phenyl of A has 1, 2, 3, 4 or 5substituents.

In some of these embodiments, the phenyl of A has 1 or 2 substituents.

It may be preferred in some of these some of these embodiments that R¹⁻⁶and n and p are such that the compound is of formula Ia, Ib or Ic.

It may be preferred in these embodiments that the optional substituentsare independently selected from the following: C₁₋₄ alkyl; C₁₋₄fluoroalkyl; C₃₋₆cycloalkyl; C₅₋₆ heteroaryl; C₅₋₆ heteroaryl methyl;C₄₋₆ heterocyclyl; C₄₋₆ heterocyclyl methyl; phenyl; benzyl; halo;amido; amidomethyl; acylamido; acylamidomethyl; C₁₋₄ alkyl ester; C₁₋₄alkyl ester methyl; C₁₋₄ alkyl carbamoyl; C₁₋₄ alkyl carbamoyl methyl;C₁₋₄ alkylacyl; C₁₋₄ alkyl acyl methyl; phneylcarbonyl; carboxy;carboxymethyl; ether; amino; aminomethyl; sulfonamido; sulfonamino;sulfone; nitrile; and nitrilemethyl.

It may be preferred in these embodiments that the optional substituentsare selected from: C₁₋₄ alkyl, fluoro, chloro, bromo, acetyl, methoxy,ethoxy, —C(═O)Me, —C(═O)Et, —CH₂C(═O)Me, phenyl, —CF₃, —CF₂H, —CN,—CH₂CN, —OBn, —OPh, —OCF₃, —OCF₂H, —O—(C₆H₄)—CN, —COOH, —CH₂COOH,—C(═O)OMe, —C(═O)NH₂, —C(═O)NMeH, —C(═O)NMe₂, —C(═O)N^(i)PrH,—C(═O)-piperidinyl, —C(═O)-pyrrolidinyl, —C(═O)-morpholino (which may bebridged or substituted by one of two methyl groups), —C(═O)-azetidinyl,—CH₂C(═O)NH₂, —CH₂C(═O)-azetidinyl, —CH₂C(═O)NMeH, —CH₂C(═O)N^(i)PrH,—CH₂C(═O)-pyrrolidinyl, —CH₂C(═O)-morpholino, —CH₂-morpholino,—CH₂-methylpiperazinyl, —OCH₂pyridinyl, —OCH₂-methyloxadiazolyl,—CH₂-imidazolyl, —O-tetrahydropyranyl, —CH₂-tetrahydropyranyl,—NH-methylpyrazinyl, —CH₂-triazolyl, —NHSO₂Ph, —NHSO₂Me, —SO₂NMePh,—SO₂NMe₂, —SO₂NHEt, —SO₂CF₃, — γ-lactam, —CH₂NHC(═O)Me, —CH₂NHC(═O)OMe,—CH₂NHC(═O)CF₃, morpholino, —CH₂NH₂, —C(═O)Ph, —OCH₂-isoxazolyl,—NH-pyrimidinyl, pyridizinyl, pyrimidinyl, pyridinyl, pyrazolyl,methylpyrazolyl, dimethylpyrazolyl, pyrazinyl, pyridazinyl,methyloxadiazolyl, oxadiazolyl, dimethyloxadiazolyl, isoxazolyl,dimethyltriazolyl, imidazolyl, benzimidazolyl and thiadiazolyl.

It may be further preferred in these embodiments that the optionalsubstituents are selected from: ethoxy, —C(═O)-morpholino (which may bebridged or substituted by one of two methyl groups), methyloxadiazolyl,cyclopropyloxadiazolyl, t-butyloxadiazolyl, cyclopropyltriazolyl, and5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-3-yl.

It may be preferred in these embodiments that, when the optionalsubstituent is a C₅₋₆ heteroaryl group, the heteroaryl ring itself issubstituted with one or more C₁₋₄ alkyl groups.

It may be preferred in the above embodiments that 1 substituent ispresent. In may be preferred in the above embodiments that 2substituents are present.

Halo and methoxy (including CF₃O) substituents may be preferred in theortho position of the phenyl group. Ethoxy and alkyl (e.g. methyl, CF₂Hand CF₃) substituents may also be preferred in the ortho position of thephenyl group. Alkyl and C₅₋₆ heteroaryl may be preferred in the metaposition of the phenyl group. Amido and amidomethyl substituents may bepreferred in the para position of the phenyl group. Particular favouredgroups in the ortho position are ethoxy, methoxy, Cl, F and CF₂H.

In some embodiments, the phenyl group bears a halo or methoxysubstituent in the ortho position, and an amido or amidomethylsubstituent in the para position of the phenyl group.

In some embodiments, the phenyl group bears an amino substituent in themeta position.

Where the substituent on phenyl is a fused C₅₋₆ N₁-containingheterocyclic ring, A may have a core structure selected from:

Particular A groups of interest include:

Optionally Substituted Naphthyl

When A is naphthyl, it may be in any orientation, e.g. naphth-1-yl,naphth-2-yl.

In some embodiments A may be optionally substituted naphthyl.

In some of these embodiments, A is unsubstituted naphthyl.

In some of these embodiments, the naphthyl ring of A has 1, 2, 3, 4 or 5substituents.

In some of these embodiments, the naphthyl ring of A has 1 or 2substituents.

It may be preferred in some of these some of these embodiments that R¹⁻⁶and n and p are such that the compound is of formula Ia, Ib or Ic.

It may be preferred in these embodiments that the optional substituentsrefers to 0-2 substituents independently selected from the following:C₁₋₄ alkyl; C₁₋₄ fluoroalkyl; C₃₋₆ cycloalkyl; C₅₋₆ heteroaryl; C₅₋₆heteroaryl methyl; C₄₋₆ heterocyclyl; C₄₋₆ heterocyclyl methyl; phenyl;benzyl; halo; amido; amidomethyl; acylamido; acylamidomethyl; C₁₋₄ alkylester; C₁₋₄ alkyl ester methyl; C₁₋₄ alkyl carbamoyl; C₁₋₄ alkylcarbamoyl methyl; C₁₋₄ alkylacyl; C₁₋₄ alkyl acyl methyl;phenylcarbonyl; carboxy; carboxymethyl; ether; amino; aminomethyl;sulfonamido; sulfonamino; sulfone; nitrile; and nitrilemethyl.

It may be preferred in these embodiments that 1 substituent is present.In may be preferred in these embodiments that 2 substituents arepresent.

Optionally Substituted C₅₋₁₂ Heteroaryl

In some embodiments A may be an optionally substituted C₅₋₁₂ heteroarylgroup.

In some of these embodiments, A is unsubstituted C₅₋₁₂ heteroaryl group.

In some of these embodiments, the C₅₋₁₂ heteroaryl of A has 1, 2, 3, 4or 5 substituents. In some of these embodiments, the C₅₋₁₂ heteroaryl ofA has 1 or 2 substituents.

It may be preferred in these embodiments that the C₅₋₁₂ heteroaryl ringis selected from one of the following: pyridinyl, pyrimidinyl,pyrazinyl, isoxazolyl, oxazolyl, thiophenyl, thiazolyl, thiadiazolyl,pyrazolyl, pyridonyl, imidazolyl, benzimidazolyl, imidazopyridinyl andquinolinyl. The heteroatoms may be in any location in the ring, whichmay be joined to the remainder of the molecule via a ring carbon atom.It may be further preferred that the C₅₋₁₂ heteroaryl ring is eitherpyridinyl or pyrimidinyl. It may also be further preferred that theC₅₋₁₂ heteroaryl is selected from pyridyl, pyrimidinyl, oxazolyl,oxadiazolyl, pyrazolyl and thiazolyl and in particular:

Further preferred groups may include benzothiazolyl and benzimidazolyoland in particular:

It may be preferred in some of these some of these embodiments that R¹⁻⁶and n and p are such that the compound is of formula Ia, Ib or Ic.

It may be preferred in these embodiments that the optional substituentsare independently selected from the following: C₁₋₄ alkyl; C₁₋₄fluoroalkyl; C₃₋₆ cycloalkyl; C₅₋₆ heteroaryl; C₅₋₆ heteroaryl methyl;C₄₋₆ heterocyclyl; C₄₋₆ heterocyclyl methyl; phenyl; benzyl; halo;amido; amidomethyl; acylamido; acylamidomethyl; C₁₋₄ alkyl ester; C₁₋₄alkyl ester methyl; C₁₋₄ alkyl carbamoyl; C₁₋₄ alkyl carbamoyl methyl;C₁₋₄ alkylacyl; C₁₋₄ alkyl acyl methyl; phneylcarbonyl; carboxy;carboxymethyl; ether; amino; aminomethyl; sulfonamido; sulfonamino;sulfone; nitrile; and nitrilemethyl.

It may be preferred in these embodiments that the optional substituentsare selected from: C₁₋₄ alkyl; C₁₋₄ fluoroalkyl; C₅₋₆ heteroaryl, C₄₋₆heterocyclyl; phenyl; halo; and ether.

It may be preferred in these embodiments that the optional substituentare selected from; methyl, ethyl, butyl, chloro, bromo, fluoro,morpholino, pyrrolidinyl, —OBn, —OPh, phenyl, para-bromophenyl,pyrazolyl, pyrimidinyl, imidazolyl and —CF₃.

In may be preferred in these embodiments that 1 substituent is present.In may be preferred in these embodiments that 2 substituents arepresent.

Halo and methoxy substituents may be preferred in the ortho position ofa C₆ heteroaryl group, or α-position of C₅ and C₇₋₁₂ heteroaryl group.Amido and amidomethyl substituents may be preferred in the para positionof a C₆ heteroaryl group, or γ-position of C₅ and C₇₋₁₂ heteroarylgroup.

In some embodiments, a C₆ heteroaryl group bears a halo or methoxysubstituent in the ortho position, and an amido or amidomethylsubstituent in the para position.

In some embodiments, a C₆ heteroaryl group bears an amino substituent inthe meta position. In some embodiments, a C₅ or C₇₋₁₂ heteroaryl groupbears an amino substituent in the β-position.

Where the C₆ heteroaryl group is 4-pyridyl, it may bear an ethersubstituent, for example in the 3-position. In some of theseembodiments, the ether substituent may be —O—C₄₋₆ heterocyclyl, whereinthe C₄₋₆ heterocyclyl may itself bear an ester group (e.g. methoxyester). It may alternatively bear an amino substituent, for example inthe 3-position. In some of these embodiments, the amino substituent maybe —NH—C₄₋₆ heterocyclyl, wherein the C₄₋₆ heterocyclyl may itself bearan ester group (e.g. —C(═O)OMe).

Where the C₆ heteroaryl group is 2-pyridyl, it may bear an amidosubstituent, for example in the 4-position.

Where the C₆ heteroaryl group is 4-pyrimidinyl, it may bear an aminosubstituent, for example in the 3-position. In some of theseembodiments, the amino substituent may be —NH—C₄₋₆ heterocyclyl, whereinthe C₄₋₆ heterocyclyl may itself bear an acyl group (e.g. —C(═O)Me).

Where A is a C₅ heteroaryl group (e.g. oxazolyl, oxadiazolyl, pyrazolyland thiazolyl), it may bear an amino, phenyl or C₆ heteroarylsubstituent in the β-position.

In some embodiments A is selected from one of the following groups:

In some embodiments A may be selected from one of the following groups:

In further embodiments, it may be preferred that A is selected from oneof the following groups:

In further embodiments, it may be preferred that A is selected from oneof the following groups:

In other further embodiments, the A group may be selected from:

phenyl with a para-amido substituent;

phenyl with a para-amido substituent, and an ortho-ethoxy group;

pyridyl with para ether or amino group, where the ether or aminosubstituent is a C₅₋₆ heterocyclic group—in these groups, the pyridyl Nmay be in the meta position; these groups may also have (in someembodiments) an ortho-ethoxy group; and

pyridyl with a meta ether group, where the pyridyl N is in the paraposition.

In further embodiments, it may be preferred that A is selected from:

EXAMPLES

The following examples are provided solely to illustrate the presentinvention and are not intended to limit the scope of the invention, asdescribed herein.

Acronyms

For convenience, many chemical moieties are represented using well knownabbreviations, including but not limited to, methyl (Me), ethyl (Et),n-propyl (nPr), isopropyl (iPr), n-butyl (nBu), tert-butyl (tBu), phenyl(Ph), benzyl (Bn), methoxy (MeO), ethoxy (EtO), trimethylsilyl (TMS),and acetyl (Ac).

For convenience, many chemical compounds are represented using wellknown abbreviations, including but not limited to, methanol (MeOH),deuterated methanol (d₄-MeOD, CD₃OD), ethanol (EtOH), isopropanol(i-PrOH), ether or diethyl ether (Et₂O), ethyl acetate (EtOAc), aceticacid (AcOH), acetonitrile (MeCN, ACN), dichloromethane (methylenechloride, DCM), trifluoroacetic acid (TFA), N,N-dimethylformamide (DMF),tetrahydrofuran (THF), dimethylsulfoxide (DMSO), deuterated chloroform(CDCl₃), diethylamine (DEA), deuterated dimethylsulfoxide (d₆-DMSO),N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC),N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI.HCl,EDCI), meta-chloroperoxybenzoic acid (mCPBA),1,1′-bis(diphenylphosphino)ferrocene (dppf), tert-butyloxycarbonyl (Boc,BOC), 2-(trimethylsilyl)ethoxymethyl (SEM), triethylamine (Et₃N),2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), 4-dimethylaminopyridine (DMAP),N,N-diisopropylethylamine (DIEA, DIPEA),1,1′-bis(diphenylphosphino)ferrocene dichloropalladium (II)(PdCl₂(dppf)), trans-dichlorobis(triphenylphosphine)palladium(II)(PdCl₂(PPh₃)₂), tris(dibenzylideneacetone) dipalladium(0) (Pd₂(dba)₃),tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄), propylphosphonicanhydride (T3P), and 1-hydroxybenzotriazole (HOBt),hexamethylphosphoramide (HMPA), triethylamine (TEA), dichloroethane(DCE), N-bromosuccinimide (NBS), N-N′-dicyclohexylcarbodiimide (DCC),p-toluenesulfonic acid (TsOH), 1,1′-carbonyldiimidazole (CDI), carbonmonoxide (CO), dithiothreitol (DTT), fluorenylmethyloxycarbonyl (Fmoc),hydrochloric acid (HCl), methyl tert-butyl ether (MTBE), ammoniumbicarbonate (NH₄HCO₃), phosphorus(V) oxychloride (POCl₃), thionylchloride (SOCl₂).

For convenience, many further terms are represented using well knownabbreviations, including but not limited to, bovine serum albumin (BSA),degree Celsius (° C.), gram (g), hour (h), high performance liquidchromatography (HPLC), liquid chromatography and mass spectrometry(LCMS), molar (M), mass spectrometry (MS), millimole (mmol), milligram(mg), minute (min), milliliter (mL), nanomolar (nM), normal (N), nuclearmagnetic resonance (NMR), pound per square inch (psi), thin layerchromatography (TLC), preparative thin layer chromatography (prep-TLC),microliter (μL).

General Experimental Details for Intermediate Preparations and Examples1 to 3

Unless otherwise stated the following generalisations apply. ¹H NMRspectra were recorded on a Bruker Ultrashield Plus (400 MHz) or a BrukerAVANCE (400 MHz). The multiplicity of a signal is designated by thefollowing abbreviations: s, singlet; d, doublet; t, triplet; q, quartet;dd, doublet of doublets; dt, doublet of triplets; tt, triplet oftriplets; br, broad; m, multiplet. All observed coupling constants, J,are reported in Hertz.

LCMS data was generated using either an Agilent 6100 Series Single QuadLCMS (LCMS-A), an Agilent 1260 Infinity Series UPLC/MS (LCMS-B), anAgilent 1200 Series G6110A Quadrupole LCMS or Waters 2695 alliance(LCMS-C). Chlorine isotopes are reported as ³⁵Cl, Bromine isotopes arereported as either ⁷⁹Br or ⁸¹Br or both ⁷⁹Br/⁸¹Br.

LCMS Method A (LCMS-A):

Instrument: Agilent 6100 Series Single Quad LC/MS

Agilent 1200 Series HPLC

Pump: 1200 Series G1311A Quaternary pump

Autosampler: 1200 Series G1329A Thermostatted Autosampler

Detector: 1200 Series G1314B Variable Wavelength Detector

LC conditions:

Reverse Phase HPLC analysis

Column: Luna C8 (2) 5 μm 50×4.6 mm 100 Å

Column temperature: 30° C.

Injection Volume: 5 μL

Solvent A: Water 0.1% Formic Acid

Solvent B: MeCN 0.1% Formic Acid

Gradient: 5-100% solvent B over 10 min

Detection: 254 nm or 214 nm

MS conditions:

Ion Source: Quadrupole

Ion Mode: Multimode-ES

Drying gas temp: 300° C.

Vaporizer temperature: 200° C.

Capillary voltage (V): 2000 (positive)

Capillary voltage (V): 4000 (negative)

Scan Range: 100-1000

Step size: 0.1 sec

Acquisition time: 10 min

LCMS Method B (LCMS-B):

Instrument: Agilent 1260 Infinity Series UPLC/MS

Pump: 1260 Infinity G1312B Binary pump

Autosampler: 1260 Infinity G1367E 1260 HiP ALS

Detector: 1290 Infinity G4212A 1290 DAD

LC conditions:

Reverse Phase HPLC analysis

Column: Poroshell 120 EC-C18 2.7 μm 50×3.0 mm

Column temperature: 35° C.

Injection Volume: 1 μL

Solvent A: Water 0.1% Formic Acid

Solvent B: MeCN 0.1% Formic Acid

Gradient: 5-100% solvent B over 3.8 min

Detection: monitored at 254 nm and 214 nm

MS conditions:

Ion Source: Quadrupole

Ion Mode: API-ES

Drying gas temp: 350° C.

Capillary voltage (V): 3000 (positive)

Capillary voltage (V): 3000 (negative)

Scan Range: 100-1000

Step size: 0.1 sec

Acquisition time: 5 min

LCMS Method C (LCMS-C):

Instrument: Agilent 1200 Series G6110A Quadrupole

Pump: Binary pump

Detector: DAD

LC conditions:

Reverse Phase HPLC analysis

Column: Xbridge-C18, 2.5 μm, 2.1×30 mm

Column temperature: 30° C.

Injection Volume: 1-10 μL

Solvent A: Water 0.07% Formic acid

Solvent B: Methanol

Gradient: 30-95% solvent B over 3.5 min (for medium polarity samples) or10-95% solvent B over 3.7 min (for large polarity samples)

Detection: monitored at 254 nm and 214 nm

MS conditions:

Ion Source: Quadrupole

Ion Mode: ES+

Drying gas temp: 350° C.

Drying gas flow: 10 L/min

Nebulizer pressure: 35 psi

Capillary voltage (V): 3500 (positive)

Scan Range: 50-900

Or

Instrument: Waters 2695 alliance

Pump: Quaternary Pump

Detector: 2996 Photodiode Array Detector

MS model: Micromass ZQ

LC conditions:

Column: Xbridge-C18, 3.5 μm, 2.1×50 mm

Column temperature: 30° C.

Injection volume: 1-10 μL

Acquisition of wavelength: 214 nm, 254 nm

Solvent A: 0.07% HCOOH aqueous solution

Solvent B: MeOH

Run time: 8 min

Gradient: 20-95% solvent B over 5 min

Detection: 254 nm and 214 nm

MS condition:

Ion source: ES+ (or ES−) MS range: 50˜900 m/z

Capillary: 3 kV Cone: 3 V Extractor: 3 V

Drying gas flow: 600 L/hr cone: 50 L/hr

Desolvation temperature: 300° C.

Source temperature: 100° C.

Sample Preparation:

The sample was dissolved in methanol, the concentration about 0.1-1.0mg/mL, then filtered through the syringes filter with 0.22 μm.

Preparative RP-HPLC:

Agilent 1260 Infinity HPLC system

UV detection at 210 nm and 254 nm

Gradient or isocratic elution through a Phenomenex Luna C8 (2) column100 Å Axia (250×21.2 mm; particle size 5 μm)

Flow rate: 10 mL/min

Gradients are as specified in the individual examples.

Analytical thin-layer chromatography was performed on Merck silica gel60 F254 aluminium-backed plates which were visualised using fluorescencequenching under UV light or a basic KMnO₄ dip or Ninhydrin dip.

Preparative thin-layer chromatography (prep TLC) was performed usingTklst (China), grand grade: (HPTLC): 8±2 μm>80%; (TLC): 10-40 μm. Type:GF254. Compounds were visualised by UV (254 nm).

Preparative LCMS (Method A)

Instrument:

Agilent 1200 series LC

Agilent 6120 Quadrupole Mass Detector

Agilent G1968D Active Splitter

LC conditions:

Reverse Phase HPLC analysis

Column: Zorbax SB-C18 5 μm 9.4×50 mm

Injection loop volume: 900 μL

Solvent A: Water plus 0.1% TFA

Solvent B: Acetonitrile plus 0.1% TFA

Gradient: 5-100% B over 6 min, hold 100% B for 1.5 min

Flow rate: 4 mL/min

Detection: 254 nm

MS conditions:

Ion Source: Quadrupole

Ion Mode: ES

Vaporiser Temp: 200° C.

Gas Temp: 300° C.

Capillary voltage positive (V): 4000

Capillary voltage negative (V): 4000

Scan Range: 100-700 Amu

Acquisition time: 10 min

Isocratic Pump (make-up flow):

Flow rate: 0.5 mL/min

Solvent: 50:50 water:acetonitrile plus 0.1% formic acid

Flash chromatography was performed using a Biotage Isolera purificationsystem using either Grace or RediSep® silica cartridges.

Column chromatography was performed using Tklst (China), grand grade,100-200 meshes silica gel.

Microwave irradiation was achieved using a CEM Explorer SP MicrowaveR^(2a)ctor.

Where necessary, anhydrous solvents were purchased from Sigma-Aldrich ordried using conventional methods. Solutions of inorganic acids or baseswhere made up as aqueous solutions unless stated otherwise.

Additional Cartridges used are as follows:

Phase Separator:

Manufacturer: Biotage

Product: ISOLUTE® Phase Separator (3 mL unless otherwise stated)

SCX and SCX-2 Cartridges:

Manufacturer: Biotage

Product: ISOLUTE® SCX 1 g, (6 mL SPE Column unless otherwise stated)

Manufacturer: Biotage

Product: ISOLUTE® SCX-2 1 g (6 mL Column)

Manufacturer: Silicycle

Product: SCX-2 500 mg or 5 g

Manufacturer: Agilent

Product: Bond Elut® SCX 10 g

Sample Extraction Cartridge:

Manufacturer: Waters

Product: Oasis® HLB 35 cc (6 g) LP extraction cartridge

Intermediate Preparations

(i) 4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoic acidI4

(a) Ethyl-2-ethoxy-4-methylbenzoate I1

To a mixture of 2-hydroxy-4-methylbenzoic acid (8.2 g, 53.9 mmol) andK₂CO₃ (22.4 g, 161.7 mmol) in dimethylsulfoxide (70 mL) at 40° C. wasadded and ethyl iodide (12.6 g, 80.8 mmol) dropwise over a period of 30min. The reaction was stirred for 2 h then further ethyl iodide (12.6 g,80.8 mmol) was added over 30 min. The resulting mixture was stirred afurther 8 h at 40° C., then diluted with CH₂Cl₂ (150 mL) and filtered.The filtrate was washed with water (200 mL×10) and brine (200 mL×2),dried (Na₂SO₄) and concentrated to give the desired compound as a yellowliquid (10.1 g, 90%): LCMS: RT 2.70 min; m/z 209.1 [M+H]⁺.

(b) 3-Ethoxy-4-(ethoxycarbonyl)-benzoic acid I2

To a solution of ethyl 2-ethoxy-4-methylbenzoate I1 (10.0 g, 48.1 mmol)in a mixture of pyridine (25 mL) and water (75 mL) was added KMnO₄ (22.8g, 144.2 mmol). The resulting mixture was heated at 50° C. for 48 h,then cooled and allowed to stir at room temperature for 24 h. Themixture was filtered and the filter cake washed with hot water. Thecombined aqueous filtrates were washed with EtOAc (75 mL×3) andacidified with 2M aqueous HCl solution. The mixture was extracted withCH₂Cl₂ (150 mL×3). The combined organic layers were washed with brine,dried (Na₂SO₄) and concentrated to give the desired compound as a whitesolid (5.0 g, 44%): LCMS: RT 0.25 min; m/z 239.0 [M+H]⁺.

(c) Ethyl4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoate I3

To a solution of 3-ethoxy-4-(ethoxycarbonyl)benzoic acid I2 (2.5 g, 10.4mmol) in CH₂Cl₂ (20 mL) was added 3-oxa-8-azabicyclo[3.2.1]octanehydrochloride (1.42 g, 9.5 mmol), HOBt (135.1 mg, 1.0 mmol), DIPEA (2.5g, 19.0 mmol) and EDCI (2.2 g, 11.4 mmol). The resulting mixture wasstirred at room temperature overnight. The mixture was partitioned withsaturated aqueous NaHCO₃, and extracted with CH₂Cl₂ (20 mL×2). Thecombined organic layers were washed with brine, dried (Na₂SO₄) andconcentrated. The residue was purified by column chromatography (1%methanol in dichloromethane) to give the desired compound as a yellowoil (2.5 g, 80%): LCMS: RT 2.40 min; m/z 334.1 [M+H]⁺.

(d) 4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoic acidI4

To a solution ofethyl-4-(3-oxa-8-azabicyclo-[3.2.1]-octane-8-carbonyl)-2-ethoxybenzoateI3 (2.4 g, 7.2 mmol) in a mixture of THF (20 mL), methanol (2 mL) andwater (2 mL) was added LiOH H₂O (1.5 g, 36 mmol). The resulting mixturewas stirred at room temperature for 24 h. The solvent was removed, andthe residue obtained diluted with water (20 mL). The pH of the aqueousmixture was adjusted to 6 by addition of a 2 M aqueous HCl solution.Themixture was extracted with CH₂Cl₂ (20 mL×3) and the combined organiclayers washed with brine (10 mL×2), dried (Na₂SO₄) and concentrated togive the desired compound as a yellow oil (1.7 g, 79%): ¹H NMR (400 MHz,d₄-MeOD) δ 7.83 (d, J=7.8 Hz, 1H), 7.19 (d, J=1.0 Hz, 1H), 7.10 (dd,J=7.8, 1.3 Hz, 1H), 4.65 (br s, 1H), 4.20 (q, J=7.0 Hz, 2H), 3.97 (br s,1H), 3.82 (d, J=10.8 Hz, 1H), 3.72 (d, J=11.0 Hz, 2H), 3.59 (d, J=10.9Hz, 1H), 2.13-1.94 (m, 4H), 1.45 (t, J=7.0 Hz, 3H). LCMS: RT 1.20 min;m/z 306.1 [M+H]⁺.

(ii) 4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)benzoic acid I6

(a) Methyl 4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)benzoate I5

To a solution of 4-(methoxycarbonyl)benzoic acid (664 mg, 3.7 mmol) inCH₂Cl₂ (20 mL) was added, 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride(500 mg, 3.4 mmol), DIPEA (865.9 mg, 6.7 mmol), HOBt (45 mg, 0.3 mmol)and EDCI (771.4 mg, 4.0 mmol). The resulting mixture was stirred at roomtemperature overnight. The mixture was partitioned against with aqueousNaHCO₃ (20 mL×2) and the aqueous layer extracted with CH₂Cl₂ (5 mL×2).The combined organic layers were washed with brine (20 mL×2), dried(Na₂SO₄) and concentrated. The crude residue obtained was purified bycolumn chromatography (1% methanol in CH₂Cl₂) to give the desiredcompound as a white solid (786.9 mg, 85%): LCMS: RT 0.61 min; m/z 276.1[M+H]⁺.

(b) 4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)benzoic acid I6

To a solution of methyl4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)benzoate I5 (770 mg, 2.8mmol) in a mixture of THF (20 mL), methanol (2 mL) and water (2 mL) wasadded LiOH.H₂O (587.8 mg, 14.0 mmol). The resulting mixture was stirredat room temperature overnight, then the solvent removed and the residueobtained diluted with water (20 mL). The pH of the aqueous solution wasadjusted to 6 by addition of a 2 M aqueous HCl solution. The aqueouslayer was extracted with CH₂Cl₂ (20 mL×3) and the combined organiclayers washed with brine (20 mL×2), dried (Na₂SO₄) and concentrated togive the desired compound (460 mg, 63%) as a white solid: LCMS: RT 0.83min; m/z 262.1 [M+H]⁺.

(iii) tert-Butyl(S)-3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I12)

(a)(S)-2-(tert-Butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (I8)

(S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (5.00 g, 28.2 mmol)was vigorously stirred in 1,4-dioxane (100 mL) and water (50 mL). Sodiumbicarbonate (4.74 mg, 56.4 mmol) and Boc anhydride (6.77 g, 31.0 mmol)were added and the mixture was stirred vigorously at room temperature.After 17 hours the mixture was concentrated in vacuo and the residuedissolved in water (200 mL). A 30% w/v aqueous solution of sodiumhydrogen sulfate monohydrate (30 mL) was added and the mixture extractedwith chloroform (3×200 mL). The pooled organic extracts were washed withbrine, dried over sodium sulfate and concentrated in vacuo to give thedesired compound (7.50 g, 96% yield) as a thick syrup. LCMS-B: RT 3.64min; m/z 178.1 [M-Boc+2H]⁺; m/z 276.1 [M−H]⁻

(b) tert-Butyl(S)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I9)

(S)-2-(tert-Butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (I8) (7.50 g, 27.0 mmol) was dissolved in THF (150 mL) and CDI(8.77 g, 54.1 mmol) was added. The mixture was stirred for 30 minutes atroom temperature then cooled to 0° C. A solution of sodium borohydride(1.16 g, 30.5 mmol) in water (15 mL) was added dropwise. After 40minutes the mixture was quenched with acetone (25 mL) and concentratedin vacuo. The residue was partitioned between water (250 mL) and ethylacetate (200 mL). The separated aqueous phase was extracted with ethylacetate (2×250 mL), the combined organic extracts washed with 5% w/vaqueous NaHSO₄ (250 mL), brine (200 mL), dried over sodium sulfate andconcentrated in vacuo. The residue was loaded in diethyl ether (50 mL)onto a plug of basic alumina and silica (50 mL each). The plug waseluted with diethyl ether (250 mL) and the eluate evaporated to give thedesired compound (5.93 g, 83% yield) as a colourless syrup. ¹H NMR (400MHz, CDCl₃) δ 7.25-7.06 (m, 4H), 4.82-4.59 (m, 1H), 4.57-4.38 (m, 1H),4.37-4.19 (m, 1H), 3.57-3.40 (m, overlaps with trace solvent), 3.03 (dd,J=16.1, 5.7 Hz, 1H), 2.80 (d, J=16.1 Hz, 1H), 1.50 (s, 9H). LCMS-B: RT3.66 min; m/z 164.2 [M-Boc+2H]⁺, 286.2 [M+Na]⁺

(c) tert-Butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(I10)

tert-Butyl(S)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I9)(1.50 g, 5.70 mmol), DCM (25 mL) and DMSO (5 mL) were cooled to 0° C.Triethylamine (2.38 mL, 17.1 mmol) was added, followed bypyridine-sulfur trioxide complex (2.72 g, 17.1 mmol). The mixture wasstirred at 0° C. for 10 minutes then allowed to come to roomtemperature. After 2 hours, saturated sodium bicarbonate (75 mL) andwater (75 mL) were added, and the mixture extracted with diethyl ether(3×150 mL). The pooled ether extracts were washed with 1:1water:saturated aqueous NH₄Cl (200 mL), brine (200 mL), dried oversodium sulfate and concentrated in vacuo to give the desired compound asa colourless oil which was used without further purification.

(d) tert-Butyl(S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I11A) and tert-butyl(S)-3-((S)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I11B)

A solution of tert-butyl(S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (I10) (5.70 mmol@100% conversion) in i-propanol (50 mL) was cooled to 0° C. Nitromethane(1.22 mL, 22.8 mmol) and potassium fluoride (331 mg, 5.70 mmol) wereadded and the mixture stirred for 18 hours, allowing the temperature tocome to room temperature as the ice bath thawed. The mixture was dilutedwith water (200 mL) and extracted with DCM (3×200 mL). The pooled DCMextracts were washed with brine, dried over sodium sulfate andconcentrated in vacuo. Chromatography (40 g silica cartridge, 0-20%ethyl acetate/hexanes) gave two partly overlapping peaks, which weresplit into early (11A major, colourless syrup, 697 mg, 37% yield) andlate (11B minor, colourless syrup, 170 mg, 9% yield) fractions. Overall:867 mg, 47% yield.

Data for major isomer tert-butyl(S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateI11A:

¹H NMR (400 MHz, d₄-MeOD) δ 7.25-7.14 (m, 4H), 4.85-4.49 (m, 5H), 4.44(dd, J=12.6, 9.3 Hz, 1H), 4.37-3.99 (m, overlaps with solvent), 3.19(dd, J=15.9, 3.2 Hz, 1H), 2.92 (dd, J=15.8, 5.6 Hz, 1H), 1.51 (s, 9H).LCMS-B: RT 3.71 min; m/z 223.2 [M-Boc+2H]⁺, 345.2 [M+Na]⁺; m/z 321.2[M−H]⁻

Data for minor isomer tert-butyl(S)-3-((S)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateI11B:

¹H NMR (400 MHz, d₄-MeOD) δ 7.25-7.11 (m, 4H), 4.75 (d, J=16.5 Hz, 1H),4.68-4.48 (m, 4H), 4.42-4.23 (m, overlaps with residual nitromethane),3.06 (dd, J=16.3, 6.1 Hz, 1H), 2.91 (d, J=16.1 Hz, 1H), 1.50 (s, 9H).LCMS-B: RT 3.70 min; m/z 223.2 [M-Boc+2H]⁺, 345.2 [M+Na]⁺; m/z 321.2[M−H]⁻

(e) tert-Butyl(S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I11A)

Copper Catalyst Used:

tert-Butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (I10)(1.9 mmol @100% conversion), absolute ethanol (5 mL), nitromethane (1.02mL, 19.0 mmol) and the copper catalyst (91 mg, 10 mol %) (see abovefigure, prepared according to Tetrahedron: Asymmetry (2008) 2310-2315)were stirred at room temperature. After 90 hours the mixture wasconcentrated in vacuo, chromatography (40 g silica cartridge, 0-15%ethyl acetate/hexanes) gave the desired compound (352 mg, 58% yield overtwo steps). ¹H NMR (400 MHz, d₄-MeOD) δ 7.25-7.13 (m, 4H), 4.85-4.68 (m,1H), 4.65-4.49 (m, 1H), 4.49-4.39 (m, 1H), 4.36-3.96 (m, overlaps withtrace solvent), 3.19 (dd, J=15.9, 3.2 Hz, 1H), 2.92 (dd, J=15.9, 5.6 Hz,1H), 1.51 (s, 9H). LCMS-B: RT 3.25 min; m/z 321.1 [M−H]⁻

(f) tert-Butyl(S)-3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I12)

tert-Butyl(S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I11A) (1.54 g, 4.78 mmol), absolute ethanol (75 mL) and 10% Pd/C (53%wetted with water, 1.5 g) were stirred under hydrogen (balloon). After 3hours the mixture was filtered through celite, the celite was washedwith absolute ethanol (100 mL) and the combined filtrates concentratedin vacuo to give the desired compound (1.34 g, 96% yield) as a palegrey-green syrup. LCMS-B: RT 3.27 min, m/z 293.2 [M+H]⁺

Alternate Synthesis Method

(a)(S)-2-(tert-Butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (I8)

(S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (50.0 g, 282 mmol)was vigorously stirred in a mixture of 1,4-dioxane (1000 mL) and water(500 mL). Sodium bicarbonate (47.4 g, 564 mmol) and Boc anhydride (67.7g, 310 mmol) were added and the reaction was stirred vigorously at roomtemperature for 6 days. The mixture was concentrated in vacuo and theresidue dissolved in water (2000 mL). A 30% w/v aqueous solution ofsodium hydrogen sulfate monohydrate (300 mL) was added and the mixtureextracted with chloroform (3×1000 mL). The pooled organic extracts werewashed with brine, dried over sodium sulfate and concentrated in vacuoto give the desired compound (90.0 g, quantitative) as a thick syrup.LCMS-B: RT 3.64 min; m/z 178.1 [M-Boc+2H]⁺; m/z 276.1 [M−H]⁻

(b) tert-Butyl(S)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I9)

(S)-2-(tert-Butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (I8) (54.0 g, 195 mmol) was dissolved in THF (1000 mL) and CDI(63.2 g, 390 mmol) was added. The mixture was stirred for 2 hours at 30°C. then cooled to 0° C. A solution of sodium borohydride (14.7 g, 390mmol) in water (120 mL) was added dropwise. After 3 hours the mixturewas quenched with acetone (300 mL) and concentrated in vacuo. Theresidue was partitioned between water (1000 mL) and ethyl acetate (1000mL). The separated aqueous phase was extracted with ethyl acetate (4×500mL) and the combined organic extracts washed with 5% w/v aqueous NaHSO₄(1000 mL), brine (500 mL), dried over sodium sulfate and concentrated invacuo. The residue was purified by chromatography (5-20% ethylacetate/petroleum ether) to give the desired compound (30.4 g, 59%yield) as a yellow syrup. ¹H NMR (400 MHz, CDCl₃) δ 7.25-7.06 (m, 4H),4.82-4.59 (m, 1H), 4.57-4.38 (m, 1H), 4.37-4.19 (m, 1H), 3.57-3.40 (m,overlaps with trace solvent), 3.03 (dd, J=16.1, 5.7 Hz, 1H), 2.80 (d,J=16.1 Hz, 1H), 1.50 (s, 9H). LCMS-B: RT 3.66 min; m/z 164.2[M-Boc+2H]⁺, 286.2 [M+Na]⁺

(c) tert-Butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(I10)

tert-Butyl(S)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I9) (16g, 0.06 mol), DCM (250 mL) and DMSO (75 mL) were cooled to 0° C.Triethylamine (25.1 mL, 0.18 mol) was added, followed by pyridine-sulfurtrioxide complex (28.6 g, 0.18 mol). The mixture was stirred at 0° C.for 30 minutes then allowed to come to room temperature and stirred atroom temperature overnight. Saturated sodium bicarbonate (200 mL) andwater (200 mL) were added, and the mixture extracted with diethyl ether(3×300 mL). The pooled ether extracts were washed with 1:1water:saturated aqueous NH₄Cl (200 mL), dried over sodium sulfate andconcentrated in vacuo to give the desired compound (16.0 g) as an orangeoil which was used without further purification.

(e) tert-Butyl(S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I11A)

To a solution of tert-butyl(S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (I10) (0.06 mol@100% conversion) in absolute ethanol (50 mL) was added a solution ofthe copper catalyst (6.8 g, 20 mol %) (see above figure, preparedaccording to Tetrahedron: Asymmetry (2008) 2310-2315) in absoluteethanol (10 mL). The mixture was cooled to 0° C. and nitromethane (36.0g, 0.6 mol) was added. The reaction was stirred at 0° C. for 3 days, themixture was concentrated in vacuo and purified by chromatography (5%ethyl acetate/petroleum ether) to give the desired compound (7.5 g, 39%yield over two steps). ¹H NMR (400 MHz, d₄-MeOD) δ 7.25-7.13 (m, 4H),4.85-4.68 (m, 1H), 4.65-4.49 (m, 1H), 4.49-4.39 (m, 1H), 4.36-3.96 (m,overlaps with trace solvent), 3.19 (dd, J=15.9, 3.2 Hz, 1H), 2.92 (dd,J=15.9, 5.6 Hz, 1H), 1.51 (s, 9H). LCMS-B: RT 3.25 min; m/z 321.1 [M−H]⁻

(f) tert-Butyl(S)-3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I12)

To a solution of tert-butyl(S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I11A) (7.5 g, 23.3 mmol) in absolute ethanol (100 mL) was added 10%Pd/C (7.5 g) and the reaction was stirred under an atmosphere ofhydrogen. After 3 hours, the mixture was filtered through Celite, theCelite was washed with absolute ethanol (200 mL) and the combinedfiltrates concentrated in vacuo to give the desired compound (5.3 g, 78%yield) as a pale grey solid. LCMS-B: RT 3.27 min, m/z 293.2 [M+H]⁺

Example 14-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N—((R)-2-hydroxy-2-((S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)benzamide(1)

(a) (S)-7-Hydroxy-6,8-diiodo-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid A2

To a solution of (S)-2-amino-3-(4-hydroxy-3,5-diiodophenyl)propanoicacid A1 (3.0 g, 6.9 mmol) in concentrated aqueous HCl (25 mL) was added1,2-dimethoxyethane (2 mL) and paraformaldehyde (0.78 g, 26 mmol). Themixture was stirred vigorously and slowly heated to 72° C. After 30 min,further concentrated aqueous HCl (5 mL), 1,2-dimethoxyethane (1 mL) andparaformaldehyde (0.52 g, 17.3 mmol) were added and heating continued at72° C. for 18 h. The suspension was then cooled in an ice bath and thesolids collected by filtration, washed thoroughly with1,2-dimethoxyethane and dried under vacuum to give the title compound asa brown solid (1.7 g, 57%. LCMS: RT 2.45 min; m/z 466.8 [M+Na]⁺.

(b)(S)-2-(tert-Butoxycarbonyl)-7-hydroxy-6,8-diiodo-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid A3

To a solution of(S)-7-hydroxy-6,8-diiodo-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid A2 (1.7 g, 3.8 mmol) in 1 M aqueous NaOH (10 mL) was added asolution of (Boc)₂O (917.1 mg, 4.2 mmol) in THF (2 mL). The solution waskept between pH 9 and 11 by addition of 1 M aqueous NaOH solution. Theresulting mixture was stirred at room temperature for 6 hours. Thesolvent was removed and the residue taken up in water. The pH of thesolution was adjusted to 4-5 by addition of 30% w/v aqueous NaHSO₄. Theaqueous phase was extracted with EtOAc (3×10 mL) and the combinedorganic fractions were washed with brine (2×10 mL), dried (Na₂SO₄) andconcentrated to give the title compound as a brown solid (1.61 g, 77%).LCMS: RT 2.93 min; m/z 567.9 [M+Na]⁺.

(c)(S)-2-(tert-Butoxycarbonyl)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid A4

A mixture of(S)-2-(tert-butoxycarbonyl)-7-hydroxy-6,8-diiodo-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid A3 (500 mg, 0.92 mmol), triethylamine (204.3 mg, 2.02 mmol) andPd/C (50 mg, 10%) in methanol (6 mL) was stirred at room temperatureunder a hydrogen atmosphere for 24 hours. The reaction mixture wasfiltered through Celite and the filtrate concentrated. The crude productwas resubjected to the same conditions above for a further 24 h, thenfiltered through Celite and concentrated. The residue obtained wasdiluted with water and acidified to pH 4-5 with 1 M aqueous HCl, thenextracted with EtOAc (2×10 mL). The combined organic fractions werewashed with brine (10 mL), dried (Na₂SO₄) and concentrated to give thetitle compound as a brown oil (310.7 mg, 100%). LCMS: RT 2.48 min; m/z316.1 [M+Na]⁺.

(d) (S)-2-tert-Butyl 3-methyl7-methoxy-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate A5

To a solution of(S)-2-(tert-butoxycarbonyl)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid A4 (300 mg, 1.02 mmol) in dimethyl sulfoxide (6 mL) was added K₂CO₃(424.4 mg, 3.07 mmol) and iodomethane (435.7 mg, 3.07 mmol). Theresulting mixture was stirred at room temperature overnight. Thereaction was quenched by addition of saturated aqueous NaHCO₃ (5 mL) andthe aqueous layer extracted with EtOAc (2×8 mL). The combined organicfractions were washed with brine (2×8 mL), dried (Na₂SO₄), filtered andconcentrated. The residue obtained was purified by column chromatography(6% EtOAc/petroleum ether) to give the title compound as a yellow oil(140 mg, 43%). LCMS: RT 2.87 min; m/z 344.1 [M+Na]⁺.

(e)(S)-2-(tert-Butoxycarbonyl)-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid A6

To a solution of (S)-2-tert-butyl 3-methyl7-methoxy-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate A5 (130 mg, 0.4mmol) in a mixture of THF (5 mL), methanol (0.5 mL) and H₂O (0.5 mL) wasadded LiOH.H₂O (85 mg, 2.02 mmol). The resulting mixture was stirred atroom temperature overnight then concentrated. The residue obtained wasdiluted with water (4 mL) and adjusted to pH 3-4 by addition of 2M HCl.The aqueous phase was extracted with EtOAc (3×5 mL) and the combinedorganic layers washed with brine (2×5 mL), dried (Na₂SO₄), filtered andconcentrated to give the title compound as a pale yellow oil (250 mg,100%). LCMS: RT 2.68 min; m/z 330.1 [M+Na]⁺.

(f) (S)-tert-Butyl3-(hydroxymethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate A7

To a solution of(S)-2-(tert-butoxycarbonyl)-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid A6 (250 mg, 0.81 mmol) in THF (4 mL) was added CDI (265.5 mg, 1.6mmol). The mixture was stirred at room temperature for 2 hours, thencooled to 0° C. A solution of NaBH₄ (61.5 mg, 1.6 mmol) in water (1 mL)was added dropwise and the resulting mixture stirred at room temperaturefor 3 hours. The solvent was removed in vacuo and the residue obtainedwas partitioned between water (4 mL) and EtOAc (5 mL). The aqueous layerwas extracted with EtOAc (3×8 mL) and the combined organic fractionswashed with saturated aqueous NaHSO₄ (2×8 mL) and brine (2×8 mL), dried(Na₂SO₄) and concentrated. The residue was purified by preparative TLC(25% EtOAc/petroleum ether) to give the title compound as a pale yellowoil (128.2 mg, 54%). LCMS: RT 2.67 min; m/z 316.2 [M+Na]⁺.

(g) (S)-tert-Butyl3-formyl-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate A8

To a solution of (S)-tert-butyl3-(hydroxymethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate A7(170 mg, 0.55 mmol) in a mixture of DCM (6 mL) and DMSO (1 mL) at 0° C.was added Et₃N (184.9 mg, 1.83 mmol) and pyridine sulfur trioxide (290.8mg, 1.83 mmol). The reaction was stirred at room temperature for 3 hoursthen quenched by addition of saturated aqueous NaHCO₃ (70 mL) and water(30 mL). The aqueous layer was extracted with diethyl ether (3×70 mL)and the pooled ether extracts were washed with 0.5 N aqueous HClsolution (30 mL) and brine (20 mL), dried (Na₂SO₄), filtered andconcentrated to give the title compound as a yellow oil (189.9 mg,100%). LCMS: RT 2.71 min; no molecular mass ion detected

(h) (S)-tert-Butyl3-((R)-1-hydroxy-2-nitroethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateA9

A mixture of (1R,2R)-N1,N2-bis(4-chlorobenzyl)cyclohexane-1,2-diamine(26.4 mg, 0.073 mmol) and cupric acetate monohydrate (12.1 mg, 0.061mmol) in ethanol (2 mL) was stirred at room temperature for 0.5 hours.Then a solution of (S)-tert-butyl3-formyl-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate A8 (185 mg,0.61 mmol) in ethanol (2 mL) was added. The reaction mixture was cooledto 0° C., stirred for a further 0.5 hours then CH₃NO₂ (364.3 mg, 6.06mmol) was added. The reaction was allowed to warm to room temperatureand stirred 24 hrs. The mixture was concentrated and the residueobtained purified by preparative TLC (20% EtOAc/Petroleum ether) to givethe title compound as an off-white solid (43 mg, 19%). LCMS: RT 2.70min; m/z 375.1 [M+Na]⁺.

(i) (S)-tert-Butyl3-((R)-2-amino-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateA10

A mixture of (S)-tert-butyl3-((R)-1-hydroxy-2-nitroethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateA9 (40 mg, 0.11 mg) and 10% Pd/C (48 mg) in ethanol (4 mL) was stirredat room temperature overnight under an atmosphere of H₂. The catalystwas removed by filtration through celite and the filtrate concentrated.The residue obtained was purified by preparative TLC (50%EtOAc/petroleum ether) to give the title compound as an off-white solid(16 mg, 44%). LCMS: RT 2.32 min; m/z 323.2 [M+H]⁺.

(j) (3S)-tert-Butyl3-((1R)-2-(4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzamido)-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateA11

To a solution of (S)-tert-butyl3-((R)-2-amino-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateA10 (16 mg, 0.05 mmol) and4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoic acid I4(16.7 mg, 0.055 mmol) in DCM (2.5 mL) was added, HOBt (0.67 mg, 0.005mmol), DIEA (25.6 mg, 0.199 mmol) and EDCI (19.1 mg, 0.099 mmol). Theresulting mixture was stirred at room temperature overnight thenpartitioned between saturated aqueous NaHCO₃ (5 mL) and CH₂Cl₂ (5 mL).The aqueous layer was extracted with CH₂Cl₂ (2×5 mL) and the combinedorganic fractions washed with brine (2×8 mL), dried (Na₂SO₄) andconcentrated. The residue obtained was purified by preparative TLC (40%petroleum ether/EtOAc) to give the title compound (13 mg, 43%) as awhite solid. LCMS: RT 2.86 min; m/z 632.3 [M+Na]⁺, 610.4 [M+H]⁺.

(k)4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N—((R)-2-hydroxy-2-((S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)benzamide1

A solution of (3S)-tert-butyl3-((1R)-2-(4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzamido)-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateA11 (13 mg, 0.021 mmol) in HCl/Et₂O (1.8 M, 2 mL) was stirred at roomtemperature overnight. The reaction mixture was concentrated and washedwith Et₂O (3 mL). The residue obtained was suspended in water (2 mL) andfreeze-dried to give the title compound (8 mg, 69%) as an off-whitesolid. ¹H NMR (400 MHz, CD₃OD) δ 7.98-7.96 (d, J=8.0 Hz, 1H), 7.22-7.15(m, 3H), 6.90-6.88 (m, 1H), 6.79 (s, 1H), 4.65 (s, 1H), 4.44 (q, J=7.2Hz, 2H), 4.27-4.22 (m, 3H), 3.95 (s, 1H), 3.78 (s, 3H), 3.73-3.68(m,3H), 3.63-3.60 (m, 3H), 3.24-3.08 (m, 3H), 2.07-2.00 (m, 4H), 1.43 (t,J=7.2 Hz, 3H). LCMS: RT 2.01 min; m/z 510.3 [M+H]⁺.

Example 24-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-N—((R)-2-hydroxy-2-((S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)benzamide(2)

(a) (3S)-tert-Butyl3-((1R)-2-(4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)benzamido)-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateA13

To a solution of (S)-tert-butyl3-((R)-2-amino-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateA10 (45 mg, 0.14 mmol) and4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)benzoic acid I6 (40.1 mg,0.15 mmol) in DCM (5 mL) was added HOBt (1.89 mg, 0.014 mmol), DIEA (72mg, 0.56 mmol) and EDCI (53.5 mg, 0.28 mmol). The resulting mixture wasstirred at room temperature overnight then partitioned between DCM (5mL) and saturated aqueous NaHCO₃ (5 mL). The aqueous layer was extractedwith CH₂Cl₂ (2×8 mL) and the combined organic fractions were washed withbrine (2×8 mL), dried (Na₂SO₄) and concentrated. The residue obtainedwas purified by preparative TLC (3% methanol/CH₂Cl₂) to give the titlecompound as an off-white solid (28.2 mg, 36%). LCMS: RT 2.75 min; m/z588.3 [M+Na]⁺.

(b)4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-N—((R)-2-hydroxy-2-((S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)benzamide2

A solution of (3S)-tert-butyl3-((1R)-2-(4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)benzamido)-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateA13 (25 mg, 0.044 mmol) in HCl/Et₂O (2 M, 6 mL) was stirred at roomtemperature for 6 hours. The reaction mixture was concentrated andwashed with Et₂O (3 mL). The residue obtained was taken up in water (3mL) and freeze-dried to give the title compound as an off-white solid(12 mg, 58%). ¹H NMR (400 MHz, CD₃OD) δ 7.96 (d, J=6.8 Hz, 2H), 7.60 (d,J=6.8 Hz, 2H), 7.20 (d, J=8.4 Hz, 1H), 6.89 (d, J=8.0 Hz, 1H), 6.78(s,1H), 4.65 (br s, 1H), 4.46-4.42(m, 1H), 4.35-4.27 (m, 2H), 3.94 (s, 1H),3.83-3.78 (m, 1H), 3.77 (s, 3H), 3.73-3.68 (m, 2H), 3.63-3.59 (m, 4H),3.26-3.12 (m, 2H), 2.07-2.00 (m, 4H); LCMS: RT 3.2 min; m/z 466.2[M+H]⁺.

Example 34-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N—((R)-2-hydroxy-2-((S)-2-methyl-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)benzamide2,2,2-trifluoroacetate (3)

(a) tert-Butyl(3S)-3-((1R)-2-(4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateA14

To a mixture of tert-butyl(S)-3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateI12 (2.7 g, 9.2 mmol),4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoic acid I4(3.1 g, 10.2 mmol), HOBt (0.13 g, 0.9 mmol) and DIPEA (4.8 g, 36.9 mmol)in dichloromethane (40 mL) was added EDCI.HCl (3.6 g, 18.5 mmol). Theresulting mixture stirred overnight at room temperature. The mixture waswashed with saturated sodium bicarbonate (20 mL) and the aqueous layerextracted with dichloromethane (2×15 mL). The combined organic extractswere washed with brine, dried over sodium sulfate, filtered andconcentrated. The residue was purified by column chromatography (50%ethyl acetate/petroleum ether) to give the title compound (2.9 g, 55%)as an off-white solid. LCMS-C: RT 2.86 min, m/z 580.4 [M+H]⁺

(b)4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N—((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)benzamidehydrochloride A15

tert-Butyl(3S)-3-((1R)-2-(4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-benzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateA14 (3.74 g, 6.46 mmol) was dissolved in diethyl ether (40 mL), stirredvigorously, and HCl in diethyl ether (1.0M, 55 mL) was added. Thereaction was stirred at ambient temperature for 18 hours before anotheraddition of HCl in diethyl ether (1.0M, 90 mL). The reaction was stirredat ambient temperature for 6 hours and the solvent removed in vacuo togive a white solid (3.7 g). The flask was cooled in an ice-water bathand a 2 M aqueous NaOH solution (150 mL, 300 mmol) was added followed byEtOAc (100 mL). The flask was vigorously stirred for 1 hour until thesolid had dissolved. The layers were separated and the aqueous layer wasextracted with EtOAc (2×100 mL). The combined organic extracts werewashed with water (100 mL), brine (100 mL), then dried over Na₂SO₄,filtered and concentrated in vacuo to give the title compound (2.92 g,94% yield) as a foamy white solid. ¹H NMR (400 MHz, CH₃OD) δ 8.02 (d,J=7.91 Hz, 1H), 7.24-6.97 (m, 6H), 4.64 (s, 1H), 4.25 (q, J=6.95 Hz,2H), 4.04-3.93 (m, 3H), 3.91-3.66 (m, 5H), 3.62-3.51 (m, 2H), 3.04-2.76(m, 3H), 2.11-1.94 (m, obscured by solvent), 1.48 (t, J=6.96 Hz, 3H).

(c)4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N—((R)-2-hydroxy-2-((S)-2-methyl-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)benzamide2,2,2-trifluoroacetate (3)

Sodium triacetoxyborohydride (104 mg, 0.491 mmol, 4.7 equiv) was addedto a solution of4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N—((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)benzamideA15 (free base, 50 mg, 0.10 mmol, 1 equiv), aqueous 37% (weight)formaldehyde solution (31 μL, 0.42 mmol, 4 equiv) and glacial aceticacid (2 drops) in methanol (20 mL). The reaction was stirred at 50° C.for 19 hours. The solvent was removed in vacuo and a saturated aqueoussolution of sodium hydrogen carbonate was added. The aqueous mixture wasextracted with EtOAc (×3) and the combined organic phases washed withwater, brine, dried over MgSO₄, filtered and the solvent removed invacuo to give a colourless oil. The oil was purified by columnchromatography (12 g SiO₂ cartridge, 0-10% MeOH in EtOAc (modified bythe addition of 0.1% v/v 7 M methanolic ammonia)) to give the crudematerial as an off-white oil. A small amount (50 mg) of the crudematerial was purified by preparative LCMS (method A) to give the titlecompound as a colourless, glass-like solid (3.2 mg, 5% yield). ¹H NMR(400 MHz, MeOD) δ 7.99 (d, J=7.9 Hz, 1H), 7.38-7.25 (m, 3H), 7.26-7.11(m, 3H), 4.76-4.61 (m, 1H), 4.57-4.45 (m, 3H), 4.25 (q, J=7.0 Hz, 2H),4.01-3.52 (m, 9H), 3.49-3.24 (m, 1H), 3.10 (s, 3H), 2.16-1.88 (m, 4H),1.44 (t, J=7.0 Hz, 3H). LCMS-B: RT 2.90 min, m/z=494.3 [M+H]⁺.

Further General Schemes

Compounds of formula H4 can be formed via a two-step sequence. An amidecoupling between carboxylic acid H1 and amino alcohol H2 affordscompounds with general formula H3. Deprotection affords compounds withgeneral formula H4.

Compound H9 can be formed in the following sequence. An amide couplingbetween carboxylic acid H5 and hydrazine B1 affords hydrazide H6.Deprotection affords hydrazide H7, which can be used to make multipleintermediates. Reaction with pivaloyl chloride affords oxadiazole H8,while ester hydrolysis affords carboxylic acid H9. Compound H12 can beprepared from H7 as follows. Reacting hydrazide H7 with acid chloride B2affords cyclopropyl hydrazide H10. Reaction with POCl₃ affordsoxadiazole H11, while ester hydrolysis affords carboxylic acid H12.

Compound B7 can be prepared from carboxylic acid B3 via an amidecoupling with hydrazide B4. Ring closure with POCl₃ affords oxadiazoleB6, which ester hydrolysis affords carboxylic acid B7.

Triazole B9 can be formed in one step from hydrazine B8 and formamide.

Compounds of formula H16 can be prepared via a two-step sequence. CHinsertion between aryl bromide H13 and heterocycle H14 affords esterH15. Ester hydrolysis affords carboxylic acid H16.

Carboxylic acid B16 can be formed via a five step sequence. Acetylationof B10 affords amide B11. Deprotection affords B12, and S_(N)Ar withdichloropyrimidine B13 affords B14. Carbonylation affords ester B15,while ester hydrolysis affords carboxylic acid B16.

Carboxylic acid B22 can be formed via a four step sequence. Palladiumcatalyzed C—N coupling between amine B17 and chloropyridine B18,followed by Boc deprotection affords B19, which upon reaction withcarbonate B20 affords carbamate B21. Ester hydrolysis affords carboxylicacid B22.

Compound 4 can be formed via the following four step sequence. Amidecoupling between carboxylic acid B23 and amino alcohol A10, followed byBoc deprotection affords compound B24. Fmoc protection of B24 isachieved by reacting amine B24 with Fmoc-OSu B25 to afford B26. S_(N)Arbetween chloropyrimidine B26 and amino oxetane B27 affords compound 4.

Amino alcohol A10 can be formed via an eight step sequence. Amino acidA1 is cyclized via a Pictet-Spengler reaction to afford THIQ B28. Bocprotection followed by hydrogenation affords carboxylic acid B29.Alkylation affords ester B30, while reduction followed by oxidationaffords aldehyde B31. Nitration followed by nitro reduction affordsamino alcohol A10.

Compound B37 can be prepared via a four step sequence. Esterification ofamino acid B32 is accomplished by reaction with thionyl chloride andmethanol, which affords ester B33. Reacting compound B33 with carbonateB20 affords carbamate B35, which can be cyclized in the presence ofparaformaldehyde and acid to afford THIQ B36. Reduction in the presenceof lithium borohydride affords protected THIQ B37. Compound B37 can beused to prepare a number of intermediates as shown in the scheme below:

A Suzuki reaction with compound B37 affords alkene B38, whiledeprotection in the presence of base affords THIQ B39. Boc protectionaffords THIQ B40, while oxidation, nitration, and hydrogenation affordamino alcohol B41. Alternatively, a palladium catalyzed cyanationreaction, followed by deprotection and Boc protection affords THIQ B42.Oxidation, nitration, and nitro reduction afford amino alcohol B43.Alternatively, a palladium catalyzed insertion of B44 affords B45.Deprotection under basic conditions affords THIQ B46 and Boc protectionaffords THIQ B47. A deprotection under basic conditions, followed byoxidation, nitration, and hydrogenation afford amino alcohol B48.

Compound B57 can be made via a 10 step sequence. Esterification ofcompound B49 in the presence of thionyl chloride and methanol affordsester B50. Reaction with carbonate B20 affords carbamate B52, and ringclosure affords THIQ B53. Deprotection followed by Boc protectionaffords carboxylic acid B55. Esterification followed by reductionaffords alcohol B56. Oxidation, nitration, and hydrogenation affordamino alcohol B57.

Preparation of Further Intermediates

Intermediate 1 (see Scheme II, Compound H9)

4-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl)benzoic acid

Step 1: To a mixture of 4-(methoxycarbonyl)benzoic acid (5.0 g, 28mmol), tert-butyl hydrazinecarboxylate (4.4 g, 33 mmol), and DMAP (13.6g, 111 mmol) was added DCM (70 mL) and EDC (6.9 g, 36 mmol). Thereaction mixture was stirred at 30° C. for 2 h. The mixture was washedwith saturated aqueous sodium bicarbonate (50 mL) and water (50 mL), andthen the organic layer was dried over anhydrous magnesium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by column chromatography on silica (0-30% EtOAc/petroleumether) to afford tert-butyl2-(4-(methoxycarbonyl)benzoyl)hydrazinecarboxylate as a solid.

Step 2: A solution of tert-butyl2-(4-(methoxycarbonyl)benzoyl)hydrazinecarboxylate (8.0 g, 27 mmol) inHCl (4 M in dioxane, 30 mL) was stirred at 30° C. for 2 h. The mixturewas concentrated under reduced pressure to afford methyl4-(hydrazinecarbonyl)benzoate hydrochloride as a solid which was useddirectly in the next step without further purification.

Step 3: To a solution of methyl 4-(hydrazinecarbonyl)benzoatehydrochloride (1.0 g, 4.3 mmol) and TEA (1.3 g, 13 mmol) in DCM (20 mL)at 0° C. under an atmosphere of nitrogen was added pivaloyl chloride(0.78 g, 6.5 mmol) dropwise. The reaction was stirred at 20° C. for 20min. The mixture was quenched with water (20 mL) and extracted with DCM(3×20 mL). The combined organic layers were dried over magnesiumsulfate, filtered, and concentrated under reduced pressure. The residuewas purified by column chromatography on silica (0-20% EtOAc/petroleumether) to afford methyl 4-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl)benzoateas an oil. MS: 261 (M+1).

Step 4: To a solution of methyl4-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl)benzoate (250 mg, 0.86 mmol) inMeOH/water (10:1, 4 mL) was added lithium hydroxide hydrate (109 mg,2.59 mmol). The mixture was stirred at 30° C. for 2 h. Then the mixturewas acidified with HCl (1 N in water, 6 mL), diluted with water (10 mL),and extracted with EtOAc (3×10 mL). The combined organic layers weredried over magnesium sulfate, filtered, and concentrated under reducedpressure to afford 4-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl)benzoic acidas a solid which was used directly in the next step without furtherpurification. MS: 247 (M+1).

Intermediate 2 (see Scheme II, Compound H12)

4-(5-cyclopropyl-1,3,4-oxadiazol-2-yl)-2-ethoxybenzoic acid

Intermediate 2 was synthesized using the same procedure asintermediate 1. MS: 275 (M+1).

Intermediate 3 (see Scheme IX, Compound A10)

(S)-tert-butyl3-((R)-2-amino-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate—AlternativeSynthesis of Compound A10, in Addition to that Detailed in Example 1

Step 1: To a solution of(S)-2-amino-3-(4-hydroxy-3,5-diiodophenyl)propanoic acid (60 g, 139mmol) in concentrated HCl (800 mL) was added 1,2-dimethoxyethane (40 mL)and paraformaldehyde (15.6 g, 520 mmol). The mixture was stirredvigorously and slowly heated to 75° C. After 30 min, more concentratedHCl (400 mL), 1,2-dimethoxyethane (20 mL) and paraformaldehyde (10.4 g,0.346 mol) were added, and the mixture was heated at 75° C. for 12 h.The suspension was cooled in an ice bath and the solid was collected byfiltration. The filter cake was washed thoroughly with1,2-dimethoxyethane (100 mL) and dried under reduced pressure to afford(S)-7-hydroxy-6,8-diiodo-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid as a solid, which was used directly in the next step withoutfurther purification. MS: 446 (M+1).

Step 2: To a solution of(S)-7-hydroxy-6,8-diiodo-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (31 g, 70 mmol) in THF (200 mL) and water (100 mL) was added sodiumcarbonate (15 g, 140 mmol) slowly. Di-tert-butyl dicarbonate (32 mL, 140mmol) was added and the mixture was stirred at 25° C. for 1 h. Themixture was concentrated under reduced pressure, and then was dilutedwith MTBE (100 mL), water (200 mL), and acidified to ˜pH 3 with citricacid. The mixture was extracted with EtOAc (2×50 mL), and the combinedorganic layers were dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure to afford(S)-2-(tert-butoxycarbonyl)-7-hydroxy-6,8-diiodo-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid as a solid, which was used directly in the next step withoutfurther purification.

Step 3: To a solution of(S)-2-(tert-butoxycarbonyl)-7-hydroxy-6,8-diiodo-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (30 g, 55 mmol) in MeOH (400 mL) under an atmosphere of nitrogenwas added palladium on carbon (10 wt %, 5.9 g, 5.5 mmol). The mixturewas stirred at 30° C. under hydrogen gas (30 psi) for 24 h. The mixturewas filtered and the filtrate was concentrated under reduced pressure toafford(S)-2-(tert-butoxycarbonyl)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid as an oil. MS: 194 (M-Boc+H).

Step 4: To a solution of(S)-2-(tert-butoxycarbonyl)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (13 g, 44 mmol) in DMSO (80 mL) was added potassium carbonate (12g, 88 mmol) and iodomethane (16.4 g, 116 mmol). The mixture was stirredat 30° C. for 4 h. The reaction was diluted with water (400 mL) andextracted with EtOAc (2×200 mL). The combined organic layers were washedwith brine (50 mL), dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by columnchromatography on silica (0-30% ethyl acetate/petroleum ether) to afford(S)-2-tert-butyl 3-methyl7-hydroxy-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate as an oil. MS:308 (M+1).

Step 5: To a solution of (S)-2-tert-butyl 3-methyl7-hydroxy-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate (3.9 g, 12.7mmol) in DMSO (40 mL) was added potassium carbonate (3.5 g, 25 mmol) andiodomethane (6.3 g, 44 mmol). The mixture was stirred at 30° C. for 5 h.The reaction was diluted with water (200 mL) and extracted with EtOAc(2×100 mL). The combined organic layers were washed with brine (50 mL),dried over anhydrous sodium sulfate, filtered, and concentrated underreduced pressure. The residue was purified by column chromatography onsilica (0-30% ethyl acetate/petroleum ether) to afford (S)-2-tert-butyl3-methyl 7-methoxy-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate as anoil. MS: 322 (M+1).

Step 6: To a solution of (S)-2-tert-butyl 3-methyl7-methoxy-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate (3 g, 9.3 mmol)in THF (30 mL) was added lithium borohydride (0.41 g, 19 mmol) at 0° C.The mixture was stirred at 30° C. for 16 h. The mixture was diluted withsaturated aqueous ammonium chloride (50 mL) and extracted with EtOAc(3×50 mL). The combined organic layers were dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure. The residuewas purified by column chromatography on silica (0-40% ethylacetate/petroleum ether) to afford (S)-tert-butyl3-(hydroxymethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate asan oil. MS: 294 (M+1).

Step 7: To a solution of oxalyl chloride (1.2 mL, 13.5 mmol) in DCM (30mL) was added DMSO (1.9 mL, 27 mmol) in DCM (2 mL) at −78° C. Thesolution was stirred at −78° C. for 0.5 h, and (S)-tert-butyl3-(hydroxymethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate(2.2 g, 7.5 mmol) in DCM (5 mL) was added dropwise. The solution wasstirred at −78° C. for 0.5 h, and TEA (7.5 mL, 54 mmol) was addeddropwise. The solution was stirred at −78° C. for 0.5 h, and slowlywarmed to 10° C. and stirred for 0.5 h. The solution was quenched withwater (50 mL), and extracted with DCM (2×50 mL). The combined organiclayers were washed with HCl (0.6 M in water, 2×100 mL), dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure to afford (S)-tert-butyl3-formyl-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate as an oilwhich was used directly in the next step without further purification.MS: 236 (M-tBu+H).

Step 8: To a solution of (S)-tert-butyl3-formyl-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate (2.0 g, 6.9mmol) in ethanol (4 mL) was added(1R,2R)-N1,N2-bis(4-chlorobenzyl)cyclohexane-1,2-diamine Copper(II)acetate complex (0.75 g, 1.4 mmol) at 30° C. The solution was cooled to0° C., nitromethane (4.2 g, 69 mmol) was added, and the reaction wasstirred at 30° C. for 40 h. The mixture was concentrated under reducedpressure, and the residue was purified by column chromatography onsilica (0-50% ethyl acetate/petroleum ether) to afford (S)-tert-butyl3-((R)-1-hydroxy-2-nitroethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil. MS: 253 (M-Boc+H).

Step 9: To a solution of (S)-tert-butyl3-((R)-1-hydroxy-2-nitroethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate(500 mg, 1.42 mmol) in ethanol (20 mL) was added Raney nickel (500 mg,8.5 mmol). The solution was stirred at 20° C. under a balloon ofhydrogen gas for 16 h. The mixture was diluted with water (20 mL) andfiltered through Celite. The filtrate was concentrated under reducedpressure to afford (S)-tert-butyl3-((R)-2-amino-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil. MS: 323 (M+1).

Intermediate 4 (see Scheme X, Compound B41)

tert-butyl(3S)-3-[(1R)-2-amino-1-hydroxyethyl]-7-ethyl-3,4-dihydroisoquinoline-2(1H)-carboxylate

Step 1: To a solution of (S)-2-amino-3-(4-bromophenyl)propanoic acid (25g, 102 mmol) in MeOH (250 mL) was added SOCl₂ (22.4 mL, 307 mmol)dropwise at 25° C. The mixture was stirred at 80° C. for 15 h. Themixture was concentrated under reduced pressure to give (S)-methyl2-amino-3-(4-bromophenyl)propanoate as a solid which was used directlyin the next step without further purification. MS: 258, 260 (M, M+2).

Step 2: To a solution of (S)-methyl 2-amino-3-(4-bromophenyl)propanoate(26.4 g, 102 mmol) in DCM (250 mL) was added TEA (18.5 mL, 133 mmol) at25° C. Added 2,5-dioxopyrrolidin-1-yl methyl carbonate (21.3 g, 123mmol), and the mixture was stirred at 25° C. for 1.5 h. The mixture wasconcentrated under reduced pressure, and the residue was purified bycolumn chromatography on silica (0-30% ethyl acetate/petroleum ether) toafford (S)-methyl 3-(4-bromophenyl)-2-((methoxycarbonyl)amino)propanoateas an oil. MS: 316, 318 (M, M+2).

Step 3: To a solution of (S)-methyl3-(4-bromophenyl)-2-((methoxycarbonyl)amino)propanoate (28 g, 89 mmol)in acetic acid/sulfuric acid (3:1, v/v, 120 mL) was addedparaformaldehyde (3.3 g, 89 mmol) at 25° C. The mixture was stirred at25° C. for 1.5 h. The mixture was poured into ice water (500 mL), anddiluted with EtOAc (200 mL). The mixture was extracted with EtOAc (4×200mL). The combined organic layers were washed with saturated aqueoussodium bicarbonate (500 mL) and brine (400 mL), dried over anhydroussodium sulfate, filtered, and concentrated under reduced pressure toafford (S)-dimethyl7-bromo-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate as an oil whichwas used directly in next step. MS: 328, 330 (M, M+2).

Step 4: To a solution of (S)-dimethyl7-bromo-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate (5 g, 15 mmol) inTHF (60 mL) was added lithium borohydride (0.50 g, 23 mmol) slowly at25° C. The mixture was stirred at 25° C. for 12 h. The reaction wasdiluted with water (100 mL) and EtOAc (50 mL). The mixture was extractedwith EtOAc (3×50 mL), and the combined organic layers were dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. The residue was purified by column chromatography on silica(0-20% ethyl acetate/petroleum ether) to afford(S)-7-bromo-10,10a-dihydro-1H-oxazolo[3,4-b]isoquinolin-3(5H)-one as asolid. MS: 308, 310 (M+K, M+K+2).

Step 5: A suspension of(S)-7-bromo-10,10a-dihydro-1H-oxazolo[3,4-b]isoquinolin-3(5H)-one (200mg, 0.75 mmol), potassium trifluoro(vinyl)borate (400 mg, 3 mmol),cesium carbonate (406 mg, 3 mmol), andchloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)(59 mg, 0.075 mmol) in THF (20 mL) and water (3 mL) was placed under anitrogen atmosphere by performing vacuum/nitrogen cycles (3×). Thereaction mixture was stirred at 85° C. for 16 h. The mixture wasconcentrated under reduced pressure, and the residue was purified bycolumn chromatography on silica (0-20% ethyl acetate/petroleum ether) toafford (S)-7-vinyl-10,10a-dihydro-1H-oxazolo[3,4-b]isoquinolin-3(5H)-oneas a solid. MS: 216 (M+1).

Step 6: To a solution of(S)-7-vinyl-10,10a-dihydro-1H-oxazolo[3,4-b]isoquinolin-3(5H)-one (1.2g, 5.6 mmol) in THF (50 mL) was added sodium hydroxide (2 M in water, 66mL, 132 mmol) at 40° C. The mixture was stirred at 40° C. for 15 h toafford (S)-(7-vinyl-1,2,3,4-tetrahydroisoquinolin-3-yl)methanol whichwas used directly in the next step. MS: 190 (M+1).

Step 7: To the solution of(S)-(7-vinyl-1,2,3,4-tetrahydroisoquinolin-3-yl)methanol (1.1 g, 5.6mmol) in THF (50 mL) and sodium hydroxide (2 M in water, 66 mL, 132mmol) obtained in step 6 was added di-tert-butyl dicarbonate (3.24 mL,13.9 mmol). The mixture was stirred at 25° C. for 1 h. The organic phasewas separated, and the aqueous phase was extracted with EtOAc (3×50 mL).The combined organic layers were washed with brine (50 mL), dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. The residue was purified by column chromatography on silica(0-40% ethyl acetate/petroleum ether) to afford (S)-tert-butyl3-(hydroxymethyl)-7-vinyl-3,4-dihydroisoquinoline-2(1H)-carboxylate asan oil. MS: 190 (M-Boc+H).

Step 8: To a solution of oxalyl chloride (0.106 mL, 1.24 mmol) in DCM (3mL) was added DMSO (0.180 mL, 2.5 mmol) in DCM (1 mL) at −78° C. Thesolution was stirred at −78° C. for 0.5 h, and (S)-tert-butyl3-(hydroxymethyl)-7-vinyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (200mg, 0.69 mmol) in DCM (1 mL) was added dropwise. The solution wasstirred at −78° C. for 0.5 h, and TEA (0.7 mL, 5 mmol) was addeddropwise. The solution was stirred at −78° C. for 0.5 h, and slowlywarmed to 25° C. for 0.5 h. The solution was quenched with water (20mL), and extracted with DCM (2×30 mL). The combined organic layers werewashed with HCl (0.25 M in water, 2×30 mL), dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure to afford(S)-tert-butyl3-formyl-7-vinyl-3,4-dihydroisoquinoline-2(1H)-carboxylate as an oilwhich was used directly in the next step without further purification.

Step 9: To a solution of (S)-tert-butyl3-formyl-7-vinyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (500 mg, 1.74mmol) in ethanol (4 mL) was added(1R,2R)—N1,N2-bis(4-chlorobenzyl)cyclohexane-1,2-diamine Copper(II)acetate complex (189 mg, 0.348 mmol) in ethanol (1 mL) at 25° C. Thesolution was cooled to 0° C., and nitromethane (1.1 g, 17.4 mmol) wasadded. The reaction was stirred at 25° C. for 48 h. The mixture wasconcentrated under reduced pressure, and the residue was purified byprep-TLC (1:5 ethyl acetate/petroleum ether, R_(f)=0.3) to afford(S)-tert-butyl3-((R)-1-hydroxy-2-nitroethyl)-7-vinyl-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil.

Step 10: To a solution of (S)-tert-butyl3-((R)-1-hydroxy-2-nitroethyl)-7-vinyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(180 mg, 0.517 mmol) in ethanol (30 mL) was added Raney nickel (200 mg,3.41 mmol) under a nitrogen atmosphere. The mixture was degassed andbackfilled with hydrogen gas (3×). The resulting mixture was stirredunder hydrogen gas (15 psi) at 25° C. for 1 h. The mixture was filtered,and the filtrate was concentrated under reduced pressure to affordtert-butyl(3S)-3-[(1R)-2-amino-1-hydroxyethyl]-7-ethyl-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil which was used directly in the next step without furtherpurification. MS: 321 (M+1).

Intermediate 5 (see Scheme X, Compound 848)

(S)-tert-butyl3-((R)-2-amino-1-hydroxyethyl)-7-(difluoromethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate

Step 1: A mixture of bis(dibenzylideneacetone)palladium(0) (0.27 g, 0.47mmol), 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (0.21 g, 0.51mmol) and cesium carbonate (12.2 g, 37.3 mmol) in toluene (60 mL) wasstirred at 25° C. for 20 min under an atmosphere of argon. Added(S)-7-bromo-10,10a-dihydro-1H-oxazolo[3,4-b]isoquinolin-3(5H)-one (2.5g, 9.3 mmol) and 2,2-difluoro-1-phenylethanone (2.2 g, 14 mmol), and thereaction mixture was heated at 100° C. for 15 h. The mixture was cooledto room temperature and water (100 mL) was added. The mixture wasextracted with EtOAc (3×50 mL) and the combined organic layers weredried over sodium sulfate, filtered, and concentrated under reducedpressure. The residue was purified by column chromatography on silica(0-50% ethyl acetate/petroleum ether) to afford(S)-7-(1,1-difluoro-2-oxo-2-phenylethyl)-10,10a-dihydro-1H-oxazolo[3,4-b]isoquinolin-3(5H)-oneas a solid. MS: 344 (M+1).

Step 2: To a mixture of(S)-7-(1,1-difluoro-2-oxo-2-phenylethyl)-10,10a-dihydro-1H-oxazolo[3,4-b]isoquinolin-3(5H)-one(2 g, 6 mmol) in toluene (30 mL) was added potassium hydroxide (1.6 g,28 mmol) and water (2 mL). The mixture was heated at 110° C. for 12 h toafford (S)-(7-(difluoromethyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)methanol. The mixture was used in the next step without furtherpurification. MS: 214 (M+1).

Step 3: To a mixture of(S)-(7-(difluoromethyl)-1,2,3,4-tetrahydroisoquinolin-3-yl) methanol(1.2 g, 6 mmol) in toluene (20 mL) was added potassium hydroxide (1.6 g,28 mmol) and water (2 mL). Di-tert-butyl dicarbonate (6.4 g, 29 mmol)was added and the mixture was stirred at 16° C. for 12 h. The reactionwas diluted with water (20 mL) and the mixture was extracted with EtOAc(2×20 mL). The combined organic layers were dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure. The residuewas purified by column chromatography on silica (0-10% ethylacetate/petroleum ether) to afford (S)-tert-butyl3-(((tert-butoxycarbonyl)oxy)methyl)-7-(difluoromethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil. MS: 436 (M+Na).

Step 4: To a mixture of (S)-tert-butyl3-(((tert-butoxycarbonyl)oxy)methyl)-7-(difluoromethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(1.2 g, 2.9 mmol) in methanol (5 mL) was added potassium carbonate (0.40g, 2.9 mmol). The mixture was stirred at 16° C. for 12 h. The mixturewas filtered, and the filtrate was concentrated under reduced pressure.The residue was purified by column chromatography on silica (0-30% ethylacetate/petroleum ether) to afford (S)-tert-butyl7-(difluoromethyl)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil.

Step 5: To a solution of oxalyl chloride (0.100 mL, 1.15 mmol) in DCM(10 mL) was added DMSO (0.16 mL, 2.3 mmol) in DCM (3 mL) at −78° C. Thesolution was stirred at −78° C. for 0.5 h, and (S)-tert-butyl7-(difluoromethyl)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(200 mg, 0.64 mmol) in DCM (3 mL) was added dropwise. The solution wasstirred at −78° C. for 0.5 h, and TEA (0.641 mL, 4.60 mmol) was addeddropwise. The solution was stirred at −78° C. for 0.5 h. The solutionwas quenched with water (40 mL), and extracted with DCM (2×40 mL). Thecombined organic layers were washed with saturated sodium chloride (40mL), dried over anhydrous sodium sulfate, filtered, and concentratedunder reduced pressure to afford (S)-tert-butyl7-(difluoromethyl)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate asan oil which was used directly in the next step without furtherpurification.

Step 6: To a solution of (S)-tert-butyl7-(difluoromethyl)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(199 mg, 0.639 mmol) in ethanol (2 mL) was added(1R,2R)—N1,N2-bis(4-chlorobenzyl)cyclohexane-1,2-diamine Copper(II)acetate complex (69 mg, 0.13 mmol) in ethanol (1 mL). The solution wascooled to 0° C., and nitromethane (0.345 mL, 6.39 mmol) was added. Thereaction was stirred at 16° C. for 18 h. The mixture was concentratedunder reduced pressure, and the residue was purified by columnchromatography on silica (0-20% ethyl acetate/petroleum ether) to afford(S)-tert-butyl7-(difluoromethyl)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil. MS: 317 (M-tBu+H).

Step 7: To a solution of (S)-tert-butyl7-(difluoromethyl)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(230 mg, 0.62 mmol) in methanol (30 mL) was added Raney nickel (500 mg,8.52 mmol) under an atmosphere of nitrogen. The mixture was degassed andbackfilled with hydrogen gas (3×). The resulting mixture was stirredunder hydrogen (15 psi) at 25° C. for 1 h. The mixture was filtered, andthe filtrate was concentrated under reduced pressure to afford(S)-tert-butyl3-((R)-2-amino-1-hydroxyethyl)-7-(difluoromethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil, which was used directly in the next step without furtherpurification. MS: 343 (M+1).

Intermediate 6 (see Scheme V, Compound H16)

4-(5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-3-yl)benzoic acid

Step 1: To an oven-dried, nitrogen-cooled vial was addedbutyldi-1-adamantylphosphine (29 mg, 0.082 mmol), palladium(II) acetate(9.2 mg, 0.041 mmol), and toluene (2 mL). The reaction mixture washeated at 50° C. for 30 minutes. To the preactivated catalyst system wasadded 5,6,7,8-tetrahydroimidazo[1,5-a]pyridine (50 mg, 0.41 mmol), ethyl4-bromobenzoate (141 mg, 0.61 mmol), potassium carbonate (170 mg, 1.2mmol), and pivalic acid (48 μL, 0.41 mmol). The mixture was evacuatedand backfilled 3× with nitrogen and stirred at 105° C. for 16 h. Thereaction was cooled to room temperature, filtered, and concentratedunder reduced pressure. The residue was purified by columnchromatography on silica gel (0-15% MeOH/DCM) to afford ethyl4-(5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-3-yl)benzoate as an oil. MS:271 (M+1).

Step 2: To a solution of ethyl4-(5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-3-yl)benzoate (41 mg, 0.15mmol) dissolved in THF (1.2 mL), MeOH (0.12 mL), and water (0.12 mL) wasadded lithium hydroxide monohydrate (32 mg, 0.75 mmol). The mixture wasstirred at room temperature for 16 h. The reaction mixture was filteredand the filtrate was concentrated under reduced pressure to affordlithium 4-(5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-3-yl)benzoate as asolid. MS: 243 (M+1).

Intermediate 7 (see Scheme IV and Scheme V, Compound H16)

4-(1-cyclopropyl-1H-1,2,4-triazol-5-yl)benzoic acid

Step 1: A solution of cyclopropylhydrazine hydrochloride (4 g, 37 mmol)in formamide (14.6 mL, 368 mmol) was heated at 130° C. for 60 h. Themixture was cooled to room temperature and brine (100 mL) was added. Themixture was extracted with EtOAc (3×30 mL), and the combined organiclayers were washed with brine (20 mL), dried over anhydrous magnesiumsulfate, filtered, and concentrated under reduced pressure. The residuewas purified by column chromatography on silica (0-40% ethylacetate/petroleum ether) to afford 1-cyclopropyl-1H-1,2,4-triazole as anoil.

Step 2: A mixture of 1-cyclopropyl-1H-1,2,4-triazole (200 mg, 1.83mmol), methyl 4-bromobenzoate (591 mg, 2.75 mmol), 2,2-dimethylbutanoicacid (64 mg, 0.55 mmol), di(adamantan-1-yl)(butyl)phosphine (263 mg,0.733 mmol), potassium carbonate (1.3 g, 9.2 mmol), and palladium(II)acetate (82 mg, 0.37 mmol) in toluene (10 mL) was degassed andbackfilled with nitrogen gas (3×). The mixture was heated at 120° C. for12 h. The mixture was concentrated under reduced pressure, and theresidue was dissolved in water (10 mL) and EtOAc (10 mL). The organiclayer was separated and the aqueous layer was extracted with EtOAc (3×15mL). The combined organic layers were washed with brine (20 mL), driedover anhydrous magnesium sulfate, filtered, and concentrated underreduced pressure. The residue was purified by prep-TLC (petroleumether:EtOAc=1:1) to afford methyl4-(1-cyclopropyl-1H-1,2,4-triazol-5-yl)benzoate as an oil. MS: 244(M+1).

Step 3: To a mixture of methyl4-(1-cyclopropyl-1H-1,2,4-triazol-5-yl)benzoate (215 mg, 0.88 mmol) inMeOH (3 mL) and water (1 mL) was added lithium hydroxide hydrate (74 mg,1.8 mmol). The solution was stirred at 10° C. for 1 h. The mixture wasconcentrated under reduced pressure. The residue was diluted with waterand the pH of the solution was adjusted by the addition of HCl (1 M inwater) to ˜pH 2. The mixture was extracted with EtOAc (3×30 mL). Thecombined organic layers were washed with brine (20 mL), dried overanhydrous magnesium sulfate, filtered, and concentrated under reducedpressure to afford 4-(1-cyclopropyl-1H-1,2,4-triazol-5-yl)benzoic acidas a solid, which was used in next step without further purification.MS: 230 (M+1).

Intermediate 8 (see Scheme III, Compound B7)

2-ethoxy-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoic acid

Step 1: To a solution of 3-ethoxy-4-(ethoxycarbonyl)benzoic acid (1 g,4.2 mmol) in DCM (35 mL) was added HATU (2.1 g, 5.5 mmol), DIEA (2.2 mL,12.6 mmol), and acetohydrazide (0.311 g, 4.20 mmol). The solution wasstirred at 20° C. for 16 h, and the solution was concentrated underreduced pressure. The residue was purified by column chromatography onsilica (0-5% MeOH/DCM) to afford ethyl4-(2-acetylhydrazinecarbonyl)-2-ethoxybenzoate as a solid. MS: 295(M+1).

Step 2: A mixture of ethyl4-(2-acetylhydrazinecarbonyl)-2-ethoxybenzoate (1 g, 3.4 mmol) in POCl₃(13 mL, 140 mmol) was placed under a nitrogen atmosphere by performingthree vacuum/nitrogen cycles. The mixture was heated at 105° C. for 20h. The mixture was poured into ice water (100 mL) and extracted with DCM(2×100 mL). The combined organic layers were washed with saturatedaqueous sodium bicarbonate (70 mL), dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by column chromatography on silica (0-20% EtOAc/hexanes) toafford ethyl 2-ethoxy-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoate as asolid. MS: 277 (M+1).

Step 3: To a solution of ethyl2-ethoxy-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoate (500 mg, 1.08 mmol)in MeOH (5 mL) and water (2 mL) was added lithium hydroxide (230 mg,5.50 mmol). The reaction was stirred at 25° C. for 4 h. The reaction wasquenched by the addition of water (30 mL), and HCl (6 M in water) wasadded to ˜pH 2. The mixture was extracted with EtOAc (3×30 mL), and thecombined organic layers were washed with brine, dried over anhydrousmagnesium sulfate, filtered, and concentrated under reduced pressure toafford 2-ethoxy-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoic acid as a solidwhich was used in next step without further purification. MS: 249 (M+1).

Intermediate 9 (see Scheme XI, Compound B57)

tert-butyl(3S)-3-[(1R)-2-amino-1-hydroxyethyl]-7-(trifluoromethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate

Step 1: To a solution of(S)-2-amino-3-(4-(trifluoromethyl)phenyl)propanoic acid (7.5 g, 32 mmol)in MeOH (60 mL) was added thionyl chloride (7 mL, 96 mmol) dropwise at15° C. The reaction was stirred at 80° C. for 15 h. The mixture wasconcentrated under reduced pressure to afford (S)-methyl2-amino-3-(4-(trifluoromethyl)phenyl)propanoate as a solid which wasused directly in next step without further purification. MS: 248 (M+1).

Step 2: To a solution of (S)-methyl2-amino-3-(4-(trifluoromethyl)phenyl)propanoate (7.9 g, 32 mmol) in DCM(70 mL) was added TEA (6.7 mL, 48 mmol) and 2,5-dioxopyrrolidin-1-ylmethyl carbonate (6.7 g, 39 mmol). The reaction was stirred at 25° C.for 1 h, and the mixture was concentrated under reduced pressure. Theresidue was purified by column chromatography on silica (0-30% ethylacetate/petroleum ether) to afford (S)-methyl2-((methoxycarbonyl)amino)-3-(4-(trifluoromethyl)phenyl)propanoate as anoil. MS: 306 (M+1).

Step 3: To a solution of (S)-methyl2-((methoxycarbonyl)amino)-3-(4-(trifluoromethyl)phenyl)propanoate (300mg, 0.983 mmol) in acetic acid/sulfuric acid (3:1, v/v, 4 mL) was addedparaformaldehyde (35 mg, 0.983 mmol). The mixture was stirred at 28° C.for 1.5 h. The mixture was poured into ice water (20 mL) and dilutedwith EtOAc (10 mL). The mixture was extracted with EtOAc (4×10 mL). Thecombined organic layers were washed with saturated aqueous sodiumbicarbonate (40 mL), brine (20 mL), dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by reverse phase HPLC (ACN/water with 0.1% TFA modifier) toafford (S)-dimethyl7-(trifluoromethyl)-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate as asolid. MS: 318 (M+1).

Step 4: A mixture of (S)-dimethyl7-(trifluoromethyl)-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate (9.98g, 31.5 mmol) in concentrated HCl (50 mL) and hydrobromic acid in aceticacid (33˜40%, 50 mL) was stirred at 110° C. for 15 h. The mixture wasconcentrated under reduced pressure to afford(S)-7-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acidas a solid which was used directly in next step without furtherpurification. MS: 246 (M+1).

Step 5: To a solution of(S)-7-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(5 g, 20.4 mmol) in 1,4-dioxane (10 mL) and water (5 mL) was addedsodium carbonate (4.3 g, 41 mmol) slowly. Di-tert-butyl dicarbonate (14mL, 61 mmol) was added and the reaction was stirred at 25° C. for 1 h.The mixture was concentrated under reduced pressure, and the residue wasquenched with water (100 mL). This mixture was diluted with tert-butylmethyl ether (100 mL), and the aqueous layer was acidified to pH ˜3 withcitric acid. The mixture was extracted with EtOAc (2×100 mL), and thecombined organic layers were dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure to afford(S)-2-(tert-butoxycarbonyl)-7-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid as an oil which was used directly in next step without furtherpurification. MS: 246 (M-Boc+H).

Step 6: To a solution of(S)-2-(tert-butoxycarbonyl)-7-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (500 mg, 1.448 mmol) in toluene (10 mL) and THF (2 mL) was added(trimethylsilyl)diazomethane (1.74 mL, 3.48 mmol) slowly at 25° C. Themixture was stirred at 25° C. for 5 min. To the mixture was added AcOH(6 mL) slowly. The mixture was concentrated under reduced pressure togive (S)-2-tert-butyl 3-methyl7-(trifluoromethyl)-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate as anoil which was used directly in next step.

Step 7: To a solution of (S)-2-tert-butyl 3-methyl7-(trifluoromethyl)-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate (520mg, 1.45 mmol) in THF (6 mL) was added lithium borohydride (126 mg, 5.79mmol). The reaction was stirred at 25° C. for 15 h, and the mixture wasquenched with water (20 mL) slowly. The mixture was extracted with EtOAc(3×20 mL), and the combined organic layers were washed with brine (30mL), dried over anhydrous sodium sulfate, filtered, and concentratedunder reduced pressure. The residue was purified by columnchromatography on silica (0-30% ethyl acetate/petroleum ether) to afford(S)-tert-butyl3-(hydroxymethyl)-7-(trifluoromethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil. MS: 276 (M-tBu+H).

Step 8: To a solution of oxalyl chloride (0.093 mL, 1.1 mmol) in DCM (3mL) was added DMSO (0.154 mL, 2.17 mmol) in DCM (1 mL) at −78° C. Themixture was stirred at −78° C. for 30 min, then (S)-tert-butyl3-(hydroxymethyl)-7-(trifluoromethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(200 mg, 0.604 mmol) in DCM (2 mL) was added at −78° C. The mixture wasstirred at −78° C. for 30 min, then TEA (0.606 mL, 4.35 mmol) was addedat −78° C. The mixture was stirred at −78° C. for 20 min. The mixturewas quenched with water (20 mL) and extracted with DCM (3×20 mL). Thecombined organic layers were washed with HCl (0.1 M in water), washedwith brine (20 mL), dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure to afford (S)-tert-butyl3-formyl-7-(trifluoromethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil which was used directly in next step without furtherpurification.

Step 9: To a solution of (S)-tert-butyl3-formyl-7-(trifluoromethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(150 mg, 0.455 mmol) in ethanol (3 mL) was added(1R,2R)—N1,N2-bis(4-chlorobenzyl)cyclohexane-1,2-diamine Copper(II)acetate complex (49 mg, 0.091 mmol) in ethanol (2 mL). The solution wascooled to 0° C. and nitromethane (280 mg, 4.6 mmol) was added. Thereaction mixture was stirred at 25° C. for 48 h. The mixture wasconcentrated under reduced pressure, and the residue was purified byprep-TLC (1:3 ethyl acetate/petroleum ether, R_(f)=0.3) to afford(S)-tert-butyl3-((R)-1-hydroxy-2-nitroethyl)-7-(trifluoromethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil. MS: 335 (M-tBu+H).

Step 10: To a solution of (S)-tert-butyl3-((R)-1-hydroxy-2-nitroethyl)-7-(trifluoromethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(90 mg, 0.23 mmol) in ethanol (20 mL) was added Raney nickel (100 mg,1.70 mmol) under an atmosphere of nitrogen. The mixture was degassed andbackfilled with hydrogen (3×), and the resulting mixture was stirredunder hydrogen (15 psi) at 25° C. for 1 h. The mixture was filtered, andthe filtrate was concentrated under reduced pressure to affordtert-butyl(3S)-3-[(1R)-2-amino-1-hydroxyethyl]-7-(trifluoromethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil which was used in next step without further purification. MS:305 (M-tBu+H).

Intermediate 10 (see Scheme X, Compound B43)

tert-butyl(3S)-3-[(1R)-2-amino-1-hydroxyethyl]-7-cyano-3,4-dihydroisoquinoline-2(1H)-carboxylate

Step 1: To a mixture of(S)-7-bromo-10,10a-dihydro-1H-oxazolo[3,4-b]isoquinolin-3(5H)-one (200mg, 0.75 mmol) and dicyanozinc (438 mg, 3.73 mmol) in DMF (5 mL) wasadded Pd(PPh₃)₄ (172 mg, 0.149 mmol) at 25° C. The mixture was degassedand backfilled with nitrogen (3×), and stirred at 80° C. for 15 h underan atmosphere of nitrogen. The mixture was cooled to room temperature,quenched with water (50 mL), and extracted with EtOAc (3×20 mL). Thecombined organic layers were washed with brine (40 mL), dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. The residue was purified by prep-TLC (2:1 ethylacetate/petroleum ether, R_(f)=0.3) to afford(S)-3-oxo-3,5,10,10a-tetrahydro-1H-oxazolo[3,4-b]isoquinoline-7-carbonitrileas a solid. MS: 215 (M+1).

Step 2: To a solution of(S)-3-oxo-3,5,10,10a-tetrahydro-1H-oxazolo[3,4-b]isoquinoline-7-carbonitrile(2 g, 9.3 mmol) in THF (100 mL) was added sodium hydroxide (2M in water,187 mL, 373 mmol). The reaction mixture was stirred at 25° C. for 12 hto afford(S)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-7-carbonitrile. Thereaction was used directly in next step. MS: 189 (M+1).

Step 3: To the solution of(S)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-7-carbonitrile (2g, 10.6 mmol) in THF (100 mL) and sodium hydroxide (2M in water, 187 mL,373 mmol) obtained in step 2 was added di-tert-butyl dicarbonate (6.2mL, 27 mmol). The mixture was stirred at 25° C. for 1 h and thenextracted with EtOAc (3×50 mL). The combined organic layers were washedwith brine (50 mL), dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by columnchromatography on silica (0-50% ethyl acetate/petroleum ether) to afford(S)-tert-butyl7-cyano-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate asan oil. MS: 189 (M-Boc+H).

Step 4: To a stirred solution of oxalyl chloride (0.13 mL, 1.5 mmol) inDCM (3 mL) was added DMSO (0.213 mL, 3.00 mmol) in DCM (1 mL) at −78° C.The solution was stirred at −78° C. for 0.5 h, and (S)-tert-butyl7-cyano-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (240mg, 0.83 mmol) in DCM (1 mL) was added dropwise. The solution wasstirred at −78° C. for 0.5 h, and TEA (0.84 mL, 6 mmol) was addeddropwise. The solution was stirred at −78° C. for 0.5 h, and then slowlywarmed to room temperature and stirred for 0.5 h. The mixture wasquenched with water (30 mL) and extracted with DCM (2×20 mL). Thecombined organic layers were washed with HCl (0.25 M in water, 20 mL×2),dried over sodium sulfate, filtered, and concentrated under reducedpressure to afford (S)-tert-butyl7-cyano-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate as an oilwhich was used directly in the next step without further purification.

Step 5: To a solution of (S)-tert-butyl7-cyano-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (240 mg, 0.84mmol) in ethanol (3 mL) was added(1R,2R)—N1,N2-bis(4-chlorobenzyl)cyclohexane-1,2-diamine copper(II)acetate complex (91 mg, 0.17 mmol) in ethanol (1 mL). The solution wascooled to 0° C., and nitromethane (512 mg, 8.38 mmol) was added. Thereaction was stirred at 25° C. for 48 h, and the mixture wasconcentrated under reduced pressure. The residue was purified byprep-TLC (1:3 ethyl acetate/petroleum ether, R_(f)=0.3) to afford(S)-tert-butyl7-cyano-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil.

Step 6: To a solution of (S)-tert-butyl7-cyano-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(110 mg, 0.32 mmol) in AcOH (10 mL) was added zinc (207 mg, 3.17 mmol).The mixture was stirred at 25° C. for 12 h. More zinc (207 mg, 3.17mmol) was added, and the mixture was stirred at 25° C. for 12 h. Themixture was poured into saturated aqueous sodium bicarbonate (200 mL),and the mixture was extracted with EtOAc (3×100 mL). The combinedorganic layers were washed with brine (150 mL), dried over anhydroussodium sulfate, filtered, and concentrated under reduced pressure toafford tert-butyl(3S)-3-[(1R)-2-amino-1-hydroxyethyl]-7-cyano-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil which was used directly in next step without furtherpurification. MS: 318 (M+1).

Intermediate 11 (see Scheme VI, Compound B16)

6-((1-acetylpiperidin-4-yl)amino)pyrimidine-4-carboxylic acid

Step 1: To a solution of tert-butyl piperidin-4-ylcarbamate (10 g, 50mmol) and TEA (10 mL, 775 mmol) in DCM (50 mL) was added aceticanhydride (5.1 g, 50 mmol) at 0° C. The resulting mixture was stirred at0° C. for 1.5 h. The reaction was quenched with water (30 mL), and theorganic layer was washed with saturated aqueous sodium bicarbonate (50mL), dried over anhydrous sodium sulfate, filtered, and concentratedunder reduced pressure to afford tert-butyl(1-acetylpiperidin-4-yl)carbamate as a solid, which was used in nextstep without further purification.

Step 2: To a solution of tert-butyl (1-acetylpiperidin-4-yl)carbamate(13 g, 55 mmol) was added TFA (24 mL, 312 mmol) in DCM (25 mL). Themixture was stirred at 20° C. for 30 min. The reaction was concentratedunder reduced pressure to afford 1-(4-aminopiperidin-1-yl)ethanone, TFAas a solid, which was used in next step without further purification.

Step 3: To a solution of 1-(4-aminopiperidin-1-yl)ethanone, TFA (24 g,134 mmol) in 2-propanol (80 mL) was added DIEA (23.4 mL, 134 mmol) and4,6-dichloropyrimidine (20 g, 134 mmol). The mixture was stirred at 100°C. for 4 h under an atmosphere of nitrogen. The mixture was concentratedunder reduced pressure and the residue was dissolved in water (60 mL).The aqueous layer was extracted with EtOAc (3×80 mL), and the combinedorganic layers were washed with brine (60 mL), dried over anhydroussodium sulfate, filtered, and concentrated under reduced pressure. Theresidue was purified by column chromatography on silica (100% ethylacetate) to afford1-(4-((6-chloropyrimidin-4-yl)amino)piperidin-1-yl)ethanone as an oil.

Step 4: To a solution of1-(4-((6-chloropyrimidin-4-yl)amino)piperidin-1-yl)ethanone (10 g, 39mmol) and potassium acetate (11.6 g, 118 mmol) in ethanol (90 mL) wasadded PdCl₂(dppf) (2.9 g, 3.9 mmol). The mixture was degassed andbackfilled with CO (3×), and the resulting mixture was stirred under CO(50 psi) at 70° C. for 16 h. The mixture was concentrated under reducedpressure, and the residue was purified by column chromatography onsilica (0-5% ethyl acetate/MeOH) to afford ethyl6-((1-acetylpiperidin-4-yl)amino)pyrimidine-4-carboxylate as an oil. MS:293 (M+1).

Step 5: To a solution of ethyl6-((1-acetylpiperidin-4-yl)amino)pyrimidine-4-carboxylate (7.5 g, 26mmol) in MeOH (60 mL) and water (5 mL) was added lithium hydroxidehydrate (1.4 g, 33 mmol). The mixture was stirred at 20° C. for 1.5 h.The mixture was acidified by adding HCl (12 N in water, 2 mL), and themixture was concentrated under reduced pressure to afford6-((1-acetylpiperidin-4-yl)amino)pyrimidine-4-carboxylic acid as asolid, which was used in next step without further purification. MS: 265(M+1).

Intermediate 12 (see Scheme VII, Compound B22)

2-{[1-(methoxycarbonyl)piperidin-4-yl]amino}pyridine-4-carboxylic acid

Step 1: To a solution of tert-butyl 4-aminopiperidine-1-carboxylate (500mg, 2.5 mmol) in toluene (20 mL) was added palladium(II) acetate (56 mg,0.25 mmol), cesium carbonate (1.06 g, 3.25 mmol),2,2′-bis(diphenylphosphino)-1,1′-binaphthalene (155 mg, 0.250 mmol), andmethyl 2-chloroisonicotinate (386 mg, 2.25 mmol). The mixture was placedunder an atmosphere of nitrogen by degassing and backfilling withnitrogen (3×). The reaction mixture was stirred at 90° C. for 10 h. Themixture was diluted with water (50 mL) and extracted with DCM (3×30 mL).The combined organic layers were dried over anhydrous sodium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by column chromatography on silica (0-50% ethylacetate/petroleum ether) to afford methyl2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)isonicotinate as asolid. MS: 336 (M+1).

Step 2: A solution of methyl2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)isonicotinate (460 mg,1.37 mmol) in HCl (4 M in MeOH, 20 mL) was stirred for 2 h at 20° C. Themixture was concentrated under reduced pressure to afford methyl2-(piperidin-4-ylamino)isonicotinate dihydrochloride as a solid whichwas used directly in next step without further purification. MS: 236(M+1).

Step 3: To a solution of methyl 2-(piperidin-4-ylamino)isonicotinatedihydrochloride (351 mg, 1.14 mmol) in DCM (20 mL) at 0° C. was added2,5-dioxopyrrolidin-1-yl methyl carbonate (237 mg, 1.37 mmol) and DIEA(589 mg, 4.56 mmol). The reaction mixture was stirred at 20° C. for 2 h.The mixture was concentrated under reduced pressure and the residue waspurified by column chromatography on silica (0-50% ethylacetate/petroleum ether) to afford methyl2-((1-(methoxycarbonyl)piperidin-4-yl)amino)isonicotinate as a solid.MS: 294 (M+1).

Step 4: To a solution of methyl2-((1-(methoxycarbonyl)piperidin-4-yl)amino)isonicotinate (280 mg, 0.96mmol) in THF (2 mL), MeOH (2 mL) and water (2 mL) was added lithiumhydroxide (34 mg, 1.4 mmol) at 0° C. The reaction mixture was stirred at20° C. for 2 h. Added HCl (1 M in water) to ˜pH 3, and the mixture wasconcentrated under reduced pressure to afford2-{[1-(methoxycarbonyl)piperidin-4-yl]amino}pyridine-4-carboxylic acidas a solid, which was used in next step without further purification.MS: 280 (M+1).

Intermediate 13 (see Scheme I, Compound H3)

tert-butyl(3S)-3-[(1R)-1-hydroxy-2-{[(2-{[1-(methoxycarbonyl)piperidin-4-yl]amino}pyridin-4-yl)carbonyl]amino}ethyl]-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate

To a mixture of (S)-tert-butyl3-((R)-2-amino-1-hydroxyethyl)-6-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate(60 mg, 0.19 mmol) in DMF (2 mL) was added2-((1-(methoxycarbonyl)piperidin-4-yl)amino)isonicotinic acid (87 mg,0.31 mmol), 2,4,6-tributyl-1,3,5,2,4,6-trioxatriphosphinane2,4,6-trioxide (298 mg, 0.414 mmol) and DIEA (94 mg, 0.72 mmol). Thereaction mixture was stirred at 25° C. for 1 h. The residue was purifiedby reverse phase HPLC (ACN/water with 0.1% TFA modifier) to affordtert-butyl(3S)-3-[(1R)-1-hydroxy-2-{[(2-{[1-(methoxycarbonyl)piperidin-4-yl]amino}pyridin-4-yl)carbonyl]amino}ethyl]-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateas a solid. MS: 584 (M+1).

Example 4N—((R)-2-hydroxy-2-((S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-6-(oxetan-3-ylamino)pyrimidine-4-carboxamide

See Scheme VIII

Step 1: To a solution of 6-chloropyrimidine-4-carboxylic acid (29.5 mg,0.186 mmol), HATU (78 mg, 0.21 mmol) and DIEA (0.217 mL, 1.24 mmol) inDCM (2 mL) was added (S)-tert-butyl3-((R)-2-amino-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateA10 (50 mg, 0.16 mmol). The mixture was stirred at 25° C. for 5 min. Thereaction was purified by prep-TLC (1:2 ethyl acetate/petroleum ether,R_(f)=0.2) to afford (S)-tert-butyl3-((R)-2-(6-chloropyrimidine-4-carboxamido)-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateas a solid which was used in next step without further purification. MS:485 (M+Na).

Step 2: A mixture of (S)-tert-butyl3-((R)-2-(6-chloropyrimidine-4-carboxamido)-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate(30 mg, 0.065 mmol) in HCl (4 M in dioxane, 5 mL) was stirred at 25° C.for 30 min. The mixture was concentrated under reduced pressure toafford6-chloro-N—((R)-2-hydroxy-2-((S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)pyrimidine-4-carboxamideas a solid, which was used in next step without further purification.

Step 3: To a solution of6-chloro-N—((R)-2-hydroxy-2-((S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)pyrimidine-4-carboxamide(86 mg, 0.24 mmol) in DCM (5 mL) was added TEA (0.1 mL, 0.7 mmol) and(9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (160 mg,0.474 mmol). The mixture was stirred at 25° C. for 30 min. The mixturewas concentrated under reduced pressure, and the residue was purified byprep-TLC (1:1 ethyl acetate/petroleum ether, R_(f)=0.3) to afford(S)-(9H-fluoren-9-yl)methyl3-((R)-2-(6-chloropyrimidine-4-carboxamido)-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateas a solid. MS: 607 (M+Na).

Step 4: To a solution of (S)-(9H-fluoren-9-yl)methyl3-((R)-2-(6-chloropyrimidine-4-carboxamido)-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate(50 mg, 0.085 mmol) and DIEA (0.090 mL, 0.51 mmol) in isopropanol (5 mL)was added oxetan-3-amine (62.5 mg, 0.855 mmol). The mixture was stirredat 110° C. for 4 h. The mixture was concentrated under reduced pressure,and the residue was purified by reverse phase HPLC (ACN/water with 0.05%ammonia hydroxide modifier) to affordN—((R)-2-hydroxy-2-((S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-6-(oxetan-3-ylamino)pyrimidine-4-carboxamideas a solid. MS: 400 (M+1). ¹H NMR (400 MHz, DMSO-d₆) δ 8.69 (m, 1H),8.56-8.44 (m, 2H), 7.12 (br s, 1H), 6.98 (d, J=8.16 Hz, 1H), 6.68 (m,1H), 6.59 (s, 1H), 5.13-4.94 (m, 2H), 4.82 (t, J=6.62 Hz, 2H), 4.46 (m,2H), 4.10-3.99 (m, 1H), 3.90-3.75 (m, 2H), 3.68 (s, 3H), 3.67-3.52 (m,3H), 2.76-2.57 (m, 2H), 2.33 (br s, 1H).

Example 52-ethoxy-N-{(2R)-2-hydroxy-2-[(3S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl]ethyl}-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzamide

Step 1: To a solution of2-ethoxy-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzoic acid (Intermediate 8)(50 mg, 0.20 mmol) in DCM (2 mL) was added HATU (89 mg, 0.23 mmol) andDIEA (0.03 mL, 0.2 mmol). The reaction mixture was stirred at 30° C. for5 min. (S)-tert-butyl3-((R)-2-amino-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateA10 (60 mg, 0.186 mmol) was added to the mixture, and the reaction wasstirred at 30° C. for 2 h. The mixture was purified by prep-TLC (1:4ethyl acetate/petroleum ether) to afford (S)-tert-butyl3-((R)-2-(2-ethoxy-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzamido)-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateas an oil. MS: 553 (M+1).

Step 2: To a solution of (S)-tert-butyl3-((R)-2-(2-ethoxy-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzamido)-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate(45 mg, 0.08 mmol) in EtOAc (1 mL) was added HCl (4M in EtOAc, 4 mL).The reaction was stirred at 26° C. for 2 h. The mixture was concentratedunder reduced pressure, and the residue was purified by reverse phaseHPLC (ACN/water with 10 mM NH₄HCO₃ modifier) to afford2-ethoxy-N-{(2R)-2-hydroxy-2-[(3S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl]ethyl}-4-(5-methyl-1,3,4-oxadiazol-2-yl)benzamideas a solid. MS: 453 (M+1). ¹H NMR (400 MHz, CD₃OD) δ 8.06 (d, J=8.0 Hz,1H), 7.69 (s, 1H), 7.65 (d, J=8.0 Hz, 1H), 7.01 (d, J=8.4 Hz, 1H), 6.78(dd, J=1.6, 8.8 Hz, 1H), 6.59 (s, 1H), 4.28 (q, J=6.8 Hz, 2H), 4.05-3.92(m, 2H), 3.89-3.80 (m, 1H), 3.79-3.68 (m, 4H), 3.60-3.45 (m, 1H),2.99-2.89 (m, 1H), 2.87-2.07 (m, 2H), 2.61 (s, 3H), 1.48 (t, J=7.04 Hz,3H).

Example 64-(5-cyclopropyl-1,3,4-oxadiazol-2-yl)-2-ethoxy-N-{(2R)-2-hydroxy-2-[(3S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl]ethyl}benzamide

Step 1: To a solution of4-(5-cyclopropyl-1,3,4-oxadiazol-2-yl)-2-ethoxybenzoic acid(Intermediate 2) (70 mg, 0.26 mmol) and HATU (126 mg, 0.33 mmol) in DCM(3 mL) was added DIEA (99 mg, 0.77 mmol) and (S)-tert-butyl3-((R)-2-amino-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylateA10 (90 mg, 0.28 mmol). The reaction was stirred at 28° C. for 1 h. Themixture was dissolved in water (20 mL), and extracted with DCM (2×10mL). The combined organic layers were dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure. The residuewas purified by reverse phase HPLC (ACN/water with 0.1% TFA modifier) toafford (S)-tert-butyl3-((R)-2-(4-(5-cyclopropyl-1,3,4-oxadiazol-2-yl)-2-ethoxybenzamido)-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate,TFA as solid. MS: 579 (M+1).

Step 2: To a solution of (S)-tert-butyl3-((R)-2-(4-(5-cyclopropyl-1,3,4-oxadiazol-2-yl)-2-ethoxybenzamido)-1-hydroxyethyl)-7-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate(100 mg, 0.173 mmol) in DCM (3 mL) was added TFA (0.5 mL, 6.5 mmol). Themixture was stirred at 25° C. for 1 h. The solution was concentratedunder reduced pressure, and the residue was purified by reverse phaseHPLC (ACN/water with 10 mM NH₄HCO₃ modifier) to afford4-(5-cyclopropyl-1,3,4-oxadiazol-2-yl)-2-ethoxy-N-{(2R)-2-hydroxy-2-[(3S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl]ethyl}benzamideas a solid. MS: 479 (M+1). ¹H NMR (400 MHz, CDCl₃) δ=8.52 (m, 1H), 8.27(d, J=8.2 Hz, 1H), 7.65 (d, J=1.2 Hz, 1H), 7.57 (dd, J=1.5, 8.1 Hz, 1H),7.02 (d, J=8.4 Hz, 1H), 6.71 (dd, J=2.4, 8.4 Hz, 1H), 6.52 (d, J=2.3 Hz,1H), 4.28 (q, J=6.8 Hz, 2H), 4.09-3.94 (m, 2H), 3.93-3.78 (m, 2H), 3.75(s, 3H), 3.64-3.51 (m, 1H), 3.02 (m, 1H), 2.84-2.70 (m, 2H), 2.30-2.16(m, 1H), 1.54 (t, J=6.8 Hz, 3H), 1.27-1.12 (m, 4H).

The compounds in the following table were prepared using the methodologyherein and the general procedure described in Examples 5 and 6

Ex. Amide No. Structure and Chemical Name [M + H]⁺ Coupling Deprotection7

451 HATU HCl 8

447 HATU TFA 9

434 HATU HCl 10

464 HATU HCl 11

486 T3P HCl 12

328 T3P HCl 13

372 T3P HCl 14

361 T3P HCl 15

507 HATU HCl 16

467 T3P HCl 17

464 T3P HCl 18

484 T3P HCl

Assays

PRMT5 Biochemical Assay

Compounds of the Invention may be Tested for In Vitro Activity in theFollowing Assay:

A histone H4 derived peptide is used as substrate (amino acid sequence:Ser-Gly-Arg-Gly-Lys-Gly-Gly-Lys-Gly-Leu-Gly-Lys-Gly-Gly-Ala-Lys-Arg-His-Arg-Lys-Val-NH₂).Full-length PRMT5 enzyme (NCBI Reference sequence NP_006100.2) wasco-expressed with His₆-MEP50 in insect cells and purified via Nickelimmobilized metal affinity and gel filtration chromatography (“theenzyme”).

The 6 μL reactions are run in Greiner brand black 384-well low volumeassay plates. All reactions contained assay buffer (phosphate bufferedsaline, 0.01% (v/v) Tween-20, 0.01% (w/v) albumin from chicken eggwhite, 1 mM Dithiothreitol, 1 μM peptide substrate, 1 μM S-Adenosylmethionine, and 15 ng/reaction enzyme, with the enzyme being omittedfrom negative control reactions. Compounds were added in a volume of 100nL from dilution series prepared in DMSO, positive and negative controlreactions receiving the same volume DMSO without compound. The plateswere sealed with adhesive seals and incubated for 4 hours at 37 degreeCelsius. Reaction progress was measured using the Transcreener™ EPIGENmethyltransferase assay (BellBrook Labs, Madison, Wis.) as recommendedby the manufacturer. To each reaction 2 μL detection mix were added,containing coupling enzymes, fluorescence polarisation tracer, and AMPantibody. Plates were incubated for 90 minutes before being read on aPerkin Elmer EnVision™ plate reader in fluorescence polarisation mode.IC₅₀ values were obtained from the raw readings by calculating percentinhibition (% I) for each reaction relative to controls on the sameplate (% I=(I−CN)/(CP−CN) where CN/CP are the averages of thenegative/positive reactions, respectively), then fitting the % I datavs. compound concentration [I] to % I=(A+((B−A)/(1+((C/[I]){circumflexover ( )}D)))) where A is the lower asymptote, B is the upper asymptote,C is the IC₅₀ value, and D is the slope.

Example Number IC₅₀ (μM) 1 0.015 2 0.018 3 1.009

PRMT5 Biomarker Assay

Compounds of the Invention may be Tested for Potency to InhibitSymmetrical Dimethylation of Arginine in the Following Assay:

The cell line TE11 was seeded at a density of 6,000 cells per well in 96well optical quality tissue culture plates in DME medium and 10% foetalbovine serum, and allowed to adhere for 5 hours under standard cultureconditions (37 degree Celsius, 5% CO₂). Compound dilutions prepared inDMSO were added to the medium, with negative control wells reserved fortreatment with DMSO only and maximum inhibition controls receiving apotent PRMT5 inhibitor compound at 1 μM concentration. After incubationfor 72 hours, the cells were fixed with 3.7% formaldehyde in PBS for 30minutes at room temperature, washed with phosphate buffer saline andblocked with Odyssey blocking buffer (LI-COR, Lincoln, Nebr.). Rabbitanti-Di-Methyl Histone H4 Arginine 3 specific antibody (Epigentek) inOdyssey blocking buffer was added and incubated for 14 hours at 4 degreeCelsius. After washing, anti-rabbit secondary antibody labelled withAlexa647 dye (LifeTechnologies) and Hoechst 33342 (1 μg/mL,SigmaAldrich) were added for 1 hour incubation. Plates were washed andread on a PerkinElmer Envision 2103 in fluorescence intensity scanningmode (24 scans across the well area). The plates were imaged on aPerkinElmer Phenix high content imaging platform. Using a Columbus imageanalysis pipeline, individual nuclei were located by Hoechst 33342 stainand the methylation level was calculated from the Alexa647-relatedintensity in the same area. IC₅₀ values were obtained from the meanAlexa647-related intensity per cell by calculating percent inhibition (%I) for each well relative to controls on the same plate (%I=(I−CN)/(CP−CN) where CN/CP are the averages of the negative/maximuminhibition controls, respectively), then fitting the % I data vs.compound concentration [I] to % I=(A+((B−A)/(1+((C/[I]){circumflex over( )}D)))) where A is the lower asymptote, B is the upper asymptote, C isthe IC₅₀ value, and D is the slope.

Example Number IC₅₀ (μM) 1 0.00019 2 0.00040 3 0.00712

PRMT5-MEP50 Enzyme Methylation Assay

PRMT5/MEP50 biochemical assay is a direct measurement of the methylationactivity of the enzyme complex on a short peptide substrate derived fromthe N-terminus of H4 histone. Methylation experiment is performed withrecombinant protein. The assessment of inhibitory effect (IC₅₀) of smallmolecules is measured by the effectiveness of the compounds to inhibitthis reaction.

In this assay, the potency (IC₅₀) of each compound was determined from atwenty-point (1:2 serial dilution; top compound concentration of 100000nM) titration curve using the following outlined procedure. To each wellof a white ProxiPlus 384 well-plate, 100 nL of compound (1% DMSO infinal assay volume of 10 μL) was dispensed, followed by the addition of8 μL of 1× assay buffer (50 mM Bicine pH 8.0, 1 mM DTT, 0.004% Tween20,0.01% BSA) containing 0.5 nM of Full-length (FL)-PRMT5-MEP50 enzymecomplex (recombinant proteins from baculovirus-transfected Sf21 cells:FL-PRMT5; MW=73837 kDa and FL-MEP50; MW=38614). Plates were sealed andplaced in a 37° C. humidified chamber for 30 minutes pre-incubation withcompounds. Subsequently, each reaction was initiated by the addition of2 μL 1× assay buffer containing 75 nM biotinylated H4R3(Me1) peptide,and 15 μM S-(5′-Adenosyl)-L-Methionine Chloride (SAM). The finalreaction in each well of 10 μL consists of 0.5 nM PRMT5-MEP50,75 nMbiotinylated-peptide, and 15 μM SAM. Methylation reactions were allowedto proceed for 150 minutes in a sealed plate at 37° C. Reactions wereimmediately quenched by the addition of 1 μL of 10% formic acid. Plateswere then frozen and shipped to SAMDI™ Tech Inc. to determine thepercent conversion from K4R3(Me1) to K4R3(Me2). IC₅₀ values weredetermined by 7 parameters biphasic fit model plotting the percentproduct conversion vs. (Log₁₀) compound concentrations.

PRMT5 Cell Target Engagement (TE) Assay

The PRMT5 TE assay is a biomarker assay for identifying compounds thatinhibit symmetric dimethylation of arginine (SDMA) of PRMT5 substrates.This assay detects symmetrically dimethylated nuclear proteins usinghigh content imaging technology. Detection of the expression ofsymmetrically dimethylated nucleo proteins is through a mixture ofprimary rabbit monoclonal antibodies to SDMA (CST 13222), which in turnrecognized by an Alexafluor 488 dye-conjugated anti-rabbit IgG secondaryantibody. The IN Cell Analyzer 2200 measures nuclear Alexafluor 488fluorescent dye intensity that is directly related to the level ofexpression of symmetrically dimethylated nuclear proteins at the singlecell level. Nuclear AF488 dye intensities are compared to the mean valuefor DMSO treated cells (MIN) to report percent of inhibition for eachcompound-treated well.

In this assay, the cell potency (EC₅₀) of each compound was determinedfrom a ten point (1:3 serial dilution; top compound concentration of10000 nM) titration curve using the following outlined procedure. Eachwell of a BD falcon collagen coated black/clear bottom 384-well platewas seeded with 4000 MCF-7 cells and allowed to attach for 5 hours.Media from cell plate was removed at 0.5 mm above the bottom of theplate and replaced with 30 μL of fresh media containing 1.2× compoundsin 0.1% DMSO. Cells were treated for 3 days in 37° C. CO₂ incubator. Onday 3, cells were fixed with Cytofix, permeabilized with 0.4%Triton-X-100/Cytofix, and washed with D-PBS without Ca/Mg. Cells wereblocked with Licor Odessey blocking reagent for one hour at roomtemperature, followed by incubation with anti-SDMA (1:1000) antibody at4° C. overnight. 1° antibody was removed, followed by three washingswith DPBS without Ca/Mg and 0.05% Tween20. Hoechst (5 μg/ml), Cell Maskdeep stain (1:2000) and Alexa488-conjugated goat anti-rabbit IgG (2μg/mL) was added for 1 hour at room temperature. A final washing step(three washes) was performed before sealing plate for imaging on In CellAnalyzer 2200. Images from analyzer were uploaded to Columbus (at WP)for image analysis. IC₅₀ values were determined by 4 parameters robustfit of percent fluorescence units vs. (Log₁₀) compound concentrations.

Enzyme Methylation Assay TE Assay Ex. No. (EC₅₀ _(—) 1, nM; EC₅₀ _(—) 2,nM) (EC₅₀, nM) 1 0.27; 5    19 2 0.62; 126   17 3 47; 574  5440 4 21;8431 5 2; 890 2151 6 2; 781 203 7 3; 994 167 8 0.62; 225   80 9 0.56;334   70 10 1; 549 345 11 0.93; 451   135 12  32; 11600 4644 13 21; 80814657 14 12; 4279 3201 15 23; 7231 2187 16  7; 3697 846 17 180; 3490010000 18 0.79; 185   19

The invention claimed is:
 1. A compound of formula I:

wherein: n is 1 or 2; p is 0 or 1; R^(1a), R^(1b), R^(1c) and R^(1d) areindependently selected from the group consisting of H, halo, C₁₋₄alkoxy, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄ cycloalkyl, NH—C₁₋₄ alkyl andcyano; R^(2a) and R^(2b) are independently selected from the groupconsisting of: (i) F; (ii) H; (iii) Me; and (iv) CH₂OH; R^(2c) andR^(2d) are independently selected from the group consisting of: (i) F;(ii) H; (iii) Me; and (iv) CH₂OH; R^(2e) is H or Me; R^(3a) and R^(3b)are independently selected from H and Me; R⁴ is either H or Me; R⁵ iseither H or Me; R^(6a) and R^(6b) are independently selected from H andMe; A is either (i) optionally substituted phenyl; (ii) optionallysubstituted naphthyl; or (iii) optionally substituted C₅₋₁₂ heteroaryl;wherein when R^(2e) is H, at least one of R^(1a), R^(1b), R^(1c) andR^(1d) is selected from C₁₋₄ alkoxy, C₂₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄cycloalkyl, NH—C₁₋₄ alkyl and cyano.
 2. A compound according to claim 1,wherein n is
 1. 3. A compound according to claim 1, wherein p is
 0. 4. Acompound according to claim 1, wherein one of R^(1a), R^(1b), R^(1c) andR^(1d) is halo, C₁₋₄ alkoxy, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄cycloalkyl, NH—C₁₋₄ alkyl or cyano.
 5. A compound according to claim 1,wherein R^(1c) is methoxy.
 6. A compound according to claim 1, whereinR^(2a), R^(2b), R^(2c) and R^(2d) are all H.
 7. A compound according toclaim 1, wherein R⁴ is H.
 8. A compound according to claim 1, wherein R⁵is H.
 9. A compound according to claim 1, wherein R^(6a) and R^(6b) areboth H.
 10. A compound according to claim 1 which is of formula Ia:

wherein R^(1c) is H or methoxy and R^(2e) is H or methyl, and one ofR^(1c) and R^(2e) is not H.
 11. A compound according to claim 1, whichhas the following stereochemistry:


12. A compound according to claim 1, wherein the optional substituentson A are independently selected from the group consisting of: C₁₋₄alkyl, C₁₋₄ fluoroalkyl, C₃₋₆ cycloalkyl, C₅₋₆ heteroaryl, C₅₋₆heteroaryl methyl, C₄₋₆ heterocyclyl, C₄₋₆ heterocyclyl methyl, phenyl,benzyl, halo, amido, amidomethyl, acylamido, acylamidomethyl, C₁₋₄ alkylester, C₁₋₄ alkyl ester methyl, C₁₋₄ alkyl carbamoyl, C₁₋₄ alkylcarbamoyl methyl, C₁₋₄ alkylacyl, C₁₋₄ alkylacyl methyl, phenylcarbonyl,carboxy, carboxymethyl, ether, amino, aminomethyl, sulfonamido,sulfonamino, sulfone, sulfoxide, nitrile and nitrilemethyl and when A isphenyl, the optional substituent may also be a fused C₅₋₆ N₁-containingheterocyclic ring.
 13. A compound according to claim 12, wherein A isphenyl which has 1 or 2 substituents.
 14. A compound according to claim13, wherein the substituents are selected from: C₁₋₄ alkyl, fluoro,chloro, bromo, acetyl, methoxy, ethoxy, —C(═O)Me, —C(═O)Et, —CH₂C(═O)Me,phenyl, —CF₃, —CF₂H, —CN, —CH₂CN, —OBn, —OPh, —OCF₃, —OCF₂H,—O—(C₆H₄)—CN, —COOH, —CH₂COOH, —C(═O)OMe, —C(═O)NH₂, —C(═O)NMeH,—C(═O)NMe₂, —C(═O)N^(i)PrH, —C(═O)-piperidinyl, —C(═O)-pyrrolidinyl,—C(═O)-morpholino (which may be bridged or substituted with one or twomethyl groups), —C(═O)-azetidinyl, —CH₂C(═O)NH₂, —CH₂C(═O)-azetidinyl,—CH₂C(═O)NMeH, —CH₂C(═O)N^(i)PrH, —CH₂C(═O)-pyrrolidinyl,—CH₂C(═O)-morpholino, —CH₂-morpholino, —CH₂-methylpiperazinyl,—OCH₂pyridinyl, —OCH₂-methyloxadiazolyl, —CH₂-imidazolyl,—O-tetrahydropyranyl, —CH₂-tetraydropyanyl, —NH-methylpyrazinyl,—CH₂-triazolyl, —NHSO₂Ph, —NHSO₂Me, —SO₂NMePh, —SO₂NMe₂, —SO₂NHEt,—SO₂CF₃, -γ-lactam, —CH₂NHC(═O)Me, —CH₂NHC(═O)OMe, —CH₂NHC(═O)CF₃,morpholino, —CH₂NH₂, —C(═O)Ph, —OCH₂-isoxazolyl, —NH-pyrimidinyl,pyridizinyl, pyrimidinyl, pyridinyl, pyrazolyl, methylpyrazolyl,dimethylpyrazolyl, pyrazinyl, pyridazinyl, methyloxadiazolyl,oxadiazolyl, dimethyloxadiazolyl, isoxazolyl, dimethyltriazolyl,imidazolyl, benzimidazolyl and thiadiazolyl.
 15. A compound according toclaim 13, wherein in the ortho position of the phenyl group there is ahalo, C₁₋₄ alkyl, methoxy or ethoxy substituent.
 16. A compoundaccording to claim 13, wherein in the para position of the phenyl groupthere is an amido or amidomethyl substituent.
 17. A compound accordingto claim 1, wherein A is a C₅₋₁₂ heteroaryl group which is eitherpyridinyl or pyrimidinyl.
 18. A compound according to claim 17, whereinthe C₅₋₁₂ heteroaryl group is unsubstituted.
 19. A compound according toclaim 17, wherein the C₅₋₁₂ heteroaryl group has 1 substituent.
 20. Acompound according to claim 19, wherein in the ortho position of a C₆heteroayl group, or α-position of C₅ and C₇₋₁₂ heteroaryl group there isa halo or methoxy substituent.
 21. A compound according to claim 19,wherein in the meta position of a C₆ heteroayl group, or in theβ-position of a C₅ or C₇₋₁₂ heteroaryl group, there is an aminosubstituent.
 22. A compound according to claim 1, wherein A is selectedfrom phenyl with a para-amido substituent; phenyl with a para-amidosubstituent, and an ortho-ethoxy group; pyridyl with para ether or aminogroup, where the ether or amino substituent is a C₅₋₆ heterocyclicgroup, with an optional ortho-ethoxy group; and pyridyl with a metaether group, where the pyridyl N is in the para position.
 23. A compoundaccording to claim 1, wherein the compound is selected from the groupconsisting of:4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N—((R)-2-hydroxy-2-((S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)benzamide(1); 4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-N—((R)-2-hydroxy-2-((S)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)benzamide(2); and 4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N—((R)-2-hydroxy-2-((S)-2-methyl-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)benzamide(3).
 24. A pharmaceutical composition comprising a compound according toclaim 1, and a pharmaceutically acceptable excipient.